Expert Review of Anti-infective Therapy

ISSN: 1478-7210 (Print) 1744-8336 (Online) Journal homepage: https://www.tandfonline.com/loi/ierz20

An update on polymyxin susceptibility testing

methods for Acinetobacter baumannii

Konstantina Dafopoulou, Sophia Vourli, Athanasios Tsakris & Spyros

Pournaras

To cite this article: Konstantina Dafopoulou, Sophia Vourli, Athanasios Tsakris & Spyros
Pournaras (2019): An update on polymyxin susceptibility testing methods for Acinetobacter
baumannii, Expert Review of Anti-infective Therapy, DOI: 10.1080/14787210.2019.1667230

To link to this article: https://doi.org/10.1080/14787210.2019.1667230

Accepted author version posted online: 11

Sep 2019.

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 Publisher: Taylor & Francis & Informa UK Limited, trading as Taylor & Francis
Group

Journal: Expert Review of Anti-infective Therapy

DOI: 10.1080/14787210.2019.1667230
An update on polymyxin susceptibility testing methods for Acinetobacter
baumannii

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Konstantina Dafopoulou,1,2 Sophia Vourli,2 Athanasios Tsakris,1 and Spyros

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Pournaras*1,2

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1
Department of Microbiology, Medical School, National and Kapodistrian University
of Athens and 2Laboratory of Clinical Microbiology, Attikon University Hospital,
an
Athens, Greece
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*Corresponding author:
Spyros Pournaras
Tel: +30-210-5832353
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Fax: +30-210-5836421
E-mail: spournaras@med.uoa.gr
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 Article type: Review

An update on polymyxin susceptibility testing methods for Acinetobacter

baumannii

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Konstantina Dafopoulou,1,2 Sophia Vourli,2 Athanasios Tsakris,1 and Spyros
Pournaras*1,2

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1
Department of Microbiology, Medical School, National and Kapodistrian University
of Athens, Greece
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2
Laboratory of Clinical Microbiology, Attikon University Hospital, Athens, Greece
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*Corresponding author:
Spyros Pournaras
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National and Kapodistrian University of Athens,

Greece
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Tel: +30 210 5832353

Fax: +30 210 5834621
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E-mail: spournaras@med.uoa.gr
 Abstract

Introduction: Carbapenem-resistant Acinetobacter baumannii (CRAB) currently

comprise >50% of A. baumannii isolates in Europe and the USA and commonly are

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extensively- or pandrug-resistant. They cause severe hospital infections, which
according to CDC have very high mortality. Polymyxins are universally recognized as

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the last-line choice against CRAB infections. The role of polymyxin susceptibility
testing (ST) for these pathogens remains subject of debate although it is critical for

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selection of appropriate antimicrobial therapy. There are significant discrepancies,
derived from technical issues, between in vitro ST methods, including important
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shortcomings for diffusion and automated methods. The joint CLSI-EUCAST Working
Group has recently recommended broth microdilution (BMD) without additives as the
reference method for polymyxin ST.
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Areas covered: This review, focused on this threatening pathogen, summarizes the
current available ST methods for polymyxins and discusses the challenges encountered
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by clinical microbiological laboratories in obtaining and interpreting valid

susceptibility results. Studies on polymyxin ST methods for Acinetobacter spp.,
retrieved from Pubmed database until May 2019, were evaluated.
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Expert opinion: Currently, no single commercial ST method for polymyxins is

providing fully accurate results. Further optimization and standardization of the ST
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methods and international harmonization of guidelines should be achieved to facilitate

the accurate detection of polymyxin-resistant A. baumannii.
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Keywords: automated systems; BMD; BMD-based products; colistin resistance;

CRAB; gradient diffusion; MALDI-TOF MS; qualitative assays; selective agar media
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 Article highlights

• Polymyxins are widely used as last-line treatment options against carbapenem-

resistant A. baumannii (CRAB) infections.
• There is great need for accurate detection of polymyxin resistance, however the
optimal method in routine diagnostic laboratories is still challenging.
• There are significant discordances between different methods for polymyxin
susceptibility testing (ST), mainly due to technical issues.

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• Diffusion methods and automated systems for polymyxin ST present important
shortcomings.

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• New technologies, including qualitative assays and MALDI-TOF MS, allow
faster detection of polymyxin-resistant A. baumannii, however more research is

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required to prove their reliability in the routine practice.
• Polymyxin ST methods require further optimization and standardization in
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order to achieve accurate detection of polymyxin-resistant A. baumannii.
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 1. Introduction
Acinetobacter baumannii is increasingly responsible for severe nosocomial infections,
associated with enhanced morbidity and mortality, particularly in intensive care units
[1]. Its remarkable ability to gain resistance against most antibiotics commonly used in
clinical settings, combined with its high capacity of surviving and rapidly spreading in
the hospital environment have made it a threatening microorganism [2, 3]. Treatment
options for infections caused by extensively-drug resistant (XDR) A. baumannii are

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currently quite limited [4]. The global spread of carbapenamase-mediated resistance
has reduced the effectiveness of carbapenems in the treatment of infections by this

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pathogen [5]. Recently, the World Health Organization classified carbapenem-resistant
A. baumannii (CRAB) among the critical priority pathogens for research and

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development of new antibiotics for antibiotic-resistant bacteria [6].
One of the most potent last lines of defense against CRAB are typically the
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previously abandoned polymyxins, used as monotherapy or as components of
combination therapy [7, 8]. Colistin (polymyxin E) and polymyxin B are polycationic
bactericidal antibiotics exerting a broad-spectrum activity against Gram-negative (GN)
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bacteria by targeting the lipid A moiety of lipopolysaccharide (LPS) in the outer

membrane [9]. However, although polymyxins are still active against the majority of A.
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baumannii, polymyxin-resistant and heteroresistant clinical isolates are unfortunately

being reported with increased frequency worldwide, causing a major clinical concern
[10, 11].
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In the face of increasing prevalence of pandrug-resistant A. baumannii strains

[12], there is obvious necessity for accurate methods of polymyxin resistance
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detection. Polymyxin susceptibility testing (ST) has been subject of debate, because of
the inconsistency between different methods that results from laboratory technical
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difficulties and problems on standardization of the procedures. In this review we will

provide a comprehensive overview of the methods used for polymyxins resistance
detection and will discuss pitfalls encountered by the clinical microbiological
laboratories in polymyxin ST. For this purpose, various studies on polymyxin ST
methods for Acinetobacter spp. retrieved from Medline (via PubMed ) database until
May 2019 were evaluated. Only articles in English were included.
 2. Polymyxin ST in A. baumannii: current limitations and implications for the
clinical laboratory
Rapid and valid polymyxin ST results are extremely important for the routine clinical
microbiological laboratories in order to achieve proper therapeutic interventions and to
prevent the increase of resistance. However, the in vitro polymyxin ST presents many
problems resulting from: i) the poor diffusion of the drug in agar; ii) the polycationic
properties of the molecule that adheres to negatively charged surfaces used for MIC

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trays, such as polysterene tubes; iii) chemical heterogeneity of the drug composition
and; iv) heteroresistance of many species to colistin, including Acinetobacter [13].

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For many years, methods widely implicated for ST in routine clinical laboratories
were disk and agar gradient diffusion methods and automated systems. So far, many

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studies in the literature comparatively assessed the performance of different
commercial and standard methods for polymyxin ST, presenting discordant results.
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Τhis could be attributed to the above mentioned reasons, but mainly to the fact that
until recently there was absence of consensus from the international organizations
about the reference method for polymyxin ST leading to a controversy on the
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appropriate method for comparison [14]. To settle the matter of this problem,
EUCAST and CLSI recently set up a Polymyxin Breakpoints Working Group, which
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issued recommendations stating that the ISO-standard broth microdilution (BMD)

(20776-1) without additives is till now the only valid method [15]. Nevertheless, the
optimal method for estimating polymyxin resistance in routine diagnostic laboratories
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is still challenging. In fact, despite the EUCAST warning to use only the reference
BMD methodology [16], most laboratories still employ diffusion methods and
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automated systems for polymyxin ST in daily practice. Also recently, a number of new
commercial BMD products for colistin testing and phenotypic assays for detection of
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polymyxin resistance have been released.

2.1. Overview of currently available antimicrobial ST methods for polymyxins,

clinical breakpoints and quality control
Table 1 summarizes relevant studies evaluating the performance of various colistin and
polymyxin B ST methods for Acinetobacter spp. The evaluation of different methods
was based on the comparison of results produced by each method with those produced
by standard reference method. Only studies that applied a dilution method as reference
 standard were included. Essential agreement (EA) was defined as the percentage of
MICs that differed by one doubling dilution from the reference MIC. Categorical
agreement (CA) represented the percentage of isolates classified in the same
susceptibility category by the method under evaluation and the reference method [45].
Very major errors (VMEs) denoted false susceptibility, namely isolates susceptible
with test method and resistant with reference method, while major errors (MEs)
corresponded to false resistance [45]. There is no consensus in the literature regarding
the calculation of errors. Typically, according to ISO standard 20776-2, VMEs and

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MEs should be calculated using resistant and susceptible isolates, respectively, as the
denominator. However, several studies estimate errors putting as denominator the total

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number of isolates tested (resistant and susceptible). For these reasons and to avoid
confusion, in this paper we quote the total number of false-susceptible or false-resistant

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results and not the proportion of VMEs or MEs. According to criteria established by
ISO, a method can be considered to exhibit acceptable performance if meets the
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following: ≥90% for essential or category agreement and ≤3% for VMEs or ME [45].

2.1.1. Dilution Methods

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As already mentioned the reference method for colistin ST is MIC determination by

BMD according to the ISO standard 20776-1 [15, 46]. However, the BMD method is
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labor intensive and consequently not convenient for routine clinical microbiological
laboratories. Additionally, the interpretation of the results is not always easy,
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especially when the phenomenon of skip wells and trailing endpoints is developed,
probably due to heteroresistant A. baumannii subpopulations or the selection of
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resistant mutants during testing [47]. Another problem is that various technical issues
influence the BMD methodology. Τhe adherence of positively charged colistin to
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polysterene has been demonstrated to significantly affect the colistin BMD MIC results
[48, 49]. It has been suggested that the issue of colistin sticking to polysterene can be
lessened by the inclusion of the surfactant polysorbate-80 (P80) in BMD (BMD-P80)
[29, 50]. In particular, the addition of P80 in CAMHB decreased the binding of
polymyxins to microdilution panels and significantly reduced colistin MICs, mainly
influencing isolates with low MIC values (≤1 or ≤2 mg/L) [29, 30, 50]. Also, other
factors with an impact on polymyxin MIC BMD results are the treatment of surface or
the type of trays used [30, 49, 51]. The colistin MICs resulting from non-coated V-
 bottom polystyrene microtitre trays were shown to be significantly lower than those
obtained on tissue-culture-coated round-bottom polystyrene microtitre trays [30]. The
joint CLSI-EUCAST Working Group recently investigated the methodological issues
associated with colistin ST and recommended that the BMD methodology for colistin
should be performed in accordance with ISO, using colistin sulphate salt and untreated
polystyrene microplates without addition of P80 or other surfactants [15]. The latter
was probably based on the observation that P80 may have synergistic activity with
colistin against Gram-negative bacilli and also that the addition of surfactants did not

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ameliorate the assay’s performance [35, 52]. On the other hand, another study, which
compared BMD performed on glass-coated plates with BMD carried out on

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polystyrene-coated plates for colistin and polymyxin B, suggested that BMD using
glass-coated plates gave the most reliable results and can be considered the best

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candidate for reference standard. It is also stated that the mode and geometrical mean
of colistin MICs for polystyrene-coated plates were lower than those produced on
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glass-coated plates, and therefore the probability of excess sticking of drug to
polystyrene is rather excluded [40]. Finally, a recent study reported that nonbinding
plates can effectively prevent reductions in MICs due to adsorption of compound to the
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plate surface and allow for assays of lipophilic antibiotics without the need for added
surfactant [51]. Hence, further studies are needed to determine the extent of trays-
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based variations in polymyxin MIC determinations.

An additional factor influencing MIC determination of polymyxins is the drug
powder used for ST [13]. The probable variation that exists in composition and
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potency of colistin and polymyxin B components in formulations from different

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commercial manufacturers and batches could be a possible cause of MIC variability,

though it seems unlikely to lead in changes in category classification [13, 53–55].
Another issue is the impact of the medium on polymyxins ST. It is well known
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that the antibacterial activity of polymyxins is strongly depending on the content of

divalent cations in the medium used for ST [56]. The recommended by CLSI content
of cation concentrations in CAMHB is 20-25 mg/L for Ca++ and 10-12.5 mg/L for
Mg++, while the presence of excess Ca++ cations inhibits the activity of polymyxins
[56]. Supporting this view, recent observations have shown that Ca++ concentration in
human interstitial space fluid in vivo was about double from that obtained in CAMHB
in vitro and the antibacterial activity of colistin might be overestimated in A.
baumannii if tested in CAMHB according to CLSI recommendations [57]. However,
 further studies are needed in order to investigate the optimum content of cations in
MHB for polymyxin ST.
Until recently, agar dilution (AD) was considered the second reference MIC
method for polymyxin ST. Indeed, several studies have applied it as the reference
standard for comparison with other methods for colistin and polymyxin B ST (Table
1). The advantages of AD are the high reproducibility of the method and the ability of
testing simultaneously multiple isolates with the same assay plate [58]. Disadvantages
include that the method is time consuming and difficult to implement in clinical

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microbiological laboratories. Previously, few reports have found good concordance
between AD and BMD MIC results, even in the case of heteroresistant A. baumannii

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[22, 25]. However, other studies have shown that AD yielded considerably higher
MICs than BMD and many false polymyxin-resistant results [33, 34, 40], suggesting

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that it cannot be used reliably as the sole method for in vitro polymyxin ST, especially
in regions in where polymyxin resistance rates among A. baumannii are high [34].
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Moreover, in 2016, the joint CLSI-EUCAST Working Group advised against the use
of AD for colistin ST, until further study data have been provided [16].
The broth macrodilution method, in which the MIC determination is carried out
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in glass tubes, is very laborious and mainly employed in research laboratories. Till
now, the single study that assessed the broth macrodilution method for colistin ST in
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comparison with BMD-P80 reported high EA and no false-susceptible results [29]. A

simplified broth macrodilution method, the colistin broth disk elution (CBDE), which
overcomes many limitations associated with colistin ST, was recently reported [44].
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CBDE was performed with four 10-ml CAMHB tubes per isolate, to which 0, 1, 2, and
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4 colistin 10-μg disks were added, generating final concentrations of 0 (growth

control), 1, 2, and 4 mg/L, respectively. MICs were estimated visually. This assay,
compared with BMD, showed CA and EA of 100% on 5 colistin-susceptible and 7
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colistin-resistant A. baumannii isolates; the same excellent performance was produced

on 12 different colistin-susceptible A. baumannii isolates in comparison with broth
macrodilution and Sensititre GNX2F panels (Thermo Fisher) [44].

2.1.2. Diffusion methods

For years, the disk diffusion method is considered unreliable for polymyxin ST,
because of the poor diffusion of the large polymyxin molecule in agar. Τhe method has
 been demonstrated to yield low reproducibility and many errors compared to BMD
reference method [17, 22]. Although some studies applied modified polymyxin disk
diffusion resistant and susceptible zone diameters for Acinetobacter [17, 31, 40], in an
attempt to improve the performance of the method, EUCAST and CLSI do not
currently provide zone diameter breakpoints for polymyxins against Acinetobacter spp.
or other gram-negative organisms [59, 60]. Moreover, the joint CLSI-EUCAST
Working Group confirmed that disk diffusion using the currently available disks for
colistin (disk potencies 10, 25 and 50 µg) does not perform well. In particular, it does

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not discriminate between susceptible and resistant isolates, and thus cannot be used for
colistin ST [15, 16, 35].

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Gradient diffusion tests are an easy-to-use commercial method implemented in
routine microbiological laboratories, but concerns have been expressed regarding their

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reliability. Although some studies showed good CA between Etest (bioMérieux, Marcy
l’ Etoile, France) and BMD for polymyxin ST [19, 22, 25, 32, 40], other studies
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questioned its validity [29, 33, 35, 37, 38], considering that it may lead to inappropriate
therapy with polymyxins. At this point, it should be noted that most studies which
showed good correlation between Etest and BMD [22, 25, 32, 40] tested mainly
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polymyxin-susceptible isolates, whereas the studies that comprised adequate numbers

of polymyxin-resistant isolates, including isolates with low levels of resistance (MIC
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of 4 or 8 mg/L), produced excessive false-susceptible results when compared with

BMD [31, 33, 35, 37, 38]. On the other hand, it should be emphasized that almost all
studies evaluating the performance of Etest found that it mostly produced one or more
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twofold dilutions lower MICs than did BMD for both sensitive and resistant isolates,
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leading in unacceptable rates of EA according to the ISO requirements (Table 1) [45].

Similarly with Etest, another gradient test for colistin ST, MIC Test Strips (MTS,
Liofilchem SRL, Italy) has been found to produce lower MICs than BMD, resulting in
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a shift toward susceptibility [33, 35]. Notably, we previously evaluated the

performance of both Etest and MTS for colistin by testing 20 CRAB (18 resistant) and
found CA of 65% and 75%, respectively, with 7 and 5 colistin-resistant isolates,
respectively, falsely categorized as susceptible [33]. It is possible that the poor
correlation between gradient tests and BMD MICs is due to the poor diffusion of
polymyxins in agar [35]. It is also considered that generally gradient tests performed
slightly better for colistin-susceptible isolates (MICs ≤2 mg/L) than for colistin-
resistant ones (MICs >2 mg/L) [35]. Thus, gradient tests should be used with caution
 for colistin ST of multidrug-resistant A. baumannii with potentially elevated colistin
MIC values, although their results may be of some clinical value for highly resistant
isolates [33, 61]. Moreover, it has been suggested that gradient diffusion test results
should be confirmed by BMD, especially when colistin administration is deemed
necessary and when the resulting Etest MICs are in the range close to the susceptible
breakpoints (MIC of 1-2 mg/L), an area where most of the discrepancies were usually
observed [19, 33]. Τhe above referred shortcomings of gradient diffusion were recently
confirmed by the joint CLSI-EUCAST Working Group, which stated that the currently

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available colistin gradient tests (manufactured by bioMérieux and Liofilchem)
underestimate colistin MICs and undercall resistance, and should be withdrawn

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from use in the laboratory [16]. Also, it should be referred that gradient diffusion
methods are not U.S. FDA approved for in vitro diagnostic ST of polymyxins and must

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be used for research use only.
Another factor that worth discussion is the effect of the medium on the
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performance of gradient tests for polymyxins. The culture medium is generally one of
the most critical variables in ST methods using agar-based medium [22]. As already
mentioned, polymyxins’ activity is strongly influenced by the content of divalent
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cations in the medium [56]. So, it has been reported that cation concentration
variability of different MHA brands and lots was correlated with low accuracy and
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discrepancies in the polymyxin B MICs determined by Etest [27]. Α previous study,

which tested colistin Etest using MHA from three different manufacturers in order to
investigate if the medium could justify the high number of observed false-susceptible
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results, showed comparable findings for all three commercial MHA [29]. Furthermore,
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a recent study which tested Etest and MTS in two different in-house prepared MHA
and also the Etest on bioMerieux's Mueller Hinton E medium as recommended by the
manufacturer, observed that colistin gradient testing with all MHA produced an
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unacceptable number of false-susceptible results [35].

2.1.3. Commercial automated or semi-automated systems and BMD-based products

Various commercial BMD systems and tests are currently available for polymyxin ST.
However, no commercial polymyxin testing device is cleared by the US FDA. This is
due to the fact that FDA labels for polymyxins do not provide any breakpoints.
Moreover, most automated systems and BMD-based products have not been
 sufficiently assessed and thus the EUCAST/CLSI cannot yet recommend their use for
polymyxin ST [16].

i) Vitek2 system. The Vitek2 semi-automated system (bioMérieux) tests concentrations

for polymyxins ranging from 0.5 to 16 mg/L. Few studies have shown high CA for
polymyxins between BMD and Vitek2 [22, 33, 40]. However, most of the available
studies have reported that Vitek2 had insufficient accuracy, as it produced
unacceptably high numbers of false-susceptible results [31, 34, 38, 42]. In particular,

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the Vitek2 yielded lower MICs and had poor correlation compared with the BMD for

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isolates with MIC ≥1 mg/L [34, 42]. This discordance across the various studies may

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be attributed to the selection of isolates used for the evaluation of system performance.
Interestingly, in June 2017, bioMérieux after conducting research decided to recall the

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Vitek2 Gram-negative test kits containing colistin (cs01n), because of high rates of
erroneously susceptible results compared to AD (used as reference for cs01n
development) and compared to BMD, thus confirming the previous observations
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regarding the inaccuracy of the method [62].
Recently, a new scheme was proposed for colistin ST; it combines the Vitek2
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and a single AD screening plate supplemented with colistin 2 mg/L (Vitek2/AD
combined method). This strategy is relatively rapid and easy for implementation in
routine clinical laboratory and decreases the false-susceptible results, as the AD
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method detects resistant isolates misclassified by Vitek2 [42].

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ii) BD Phoenix system. The BD Phoenix semi-automated microbiology system (BD

Diagnostics) uses panels that test colistin concentrations of 1 to 4 mg/L. Till now, the
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single study in the literature which assessed the validity of Phoenix system for colistin
ST in comparison with BMD among A. baumannii isolates has shown worrying results.
In particular, the Phoenix100 system underestimated colistin MICs and misidentified
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12 out of 29 resistant isolates as susceptible. False susceptibility was considerably

lower among isolates displaying MICs of ≤1 mg/L compared with those yielding an
MIC of 2 mg/L, indicating that isolates with MICs within the susceptible range,
especially but not exclusively those at the susceptibility breakpoint (2 mg/L), require to
be validated by BMD [34]. Ιt should also be noted that in January 2018, BD has issued
an urgent field safety notice describing the problem of high rate of false-susceptible
results for colistin using the BD Phoenix system compared to BMD. Nevertheless, the
 company is not recommending discarding, returning or discontinuing use of BD
Phoenix Gram Negative Panels containing colistin. Consumers are advised to carry out
an alternate testing method prior to reporting Phoenix colistin-susceptible results, while
a colistin-resistant result does not require alternate testing.

iii) MicroScan WalkAway system. The Microscan system (Beckman Coulter, Inc.) tests
a limited concentration range for colistin (2 to 4 mg/L). Scarce data are available
regarding the accuracy of the MicroScan system for colistin ST. In a recent study, the

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authors referred that the MicroScan walkaway system failed to detect one out of the 8

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colistin-resistant A. baumannii isolates, while 3 out of 4 colistin-susceptible isolates

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were found falsely resistant compared to BMD, suggesting that non-susceptible results
for non-fermenters should be confirmed by another method [39]. Currently, although

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the test panels include colistin, the Microscan system does not allow the report of
colistin ST results for A. baumannii. an
iv) Sensititre system. The Sensititre system (Thermo Fisher Scientific) utilizes 96-well
microtitre plates, available in both standard and custom formats for colistin. The
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method can use an automated incubation and reading system or in case of low-volume
laboratories the plates can be inoculated and read manually. Comparison of the
Sensititre system with the BMD reference method for Acinetobacter isolates showed
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EA and CA of 91%, with error categorization for 2 out of the 14 colistin-susceptible

isolates, but there were no false-susceptible results [35]. Another study demonstrated
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excellent rate of CA (100%), and as such no false-susceptible or false-resistant results

[39].
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v) SensiTest. The SensiTest (Liofilchem) is a compact panel containing colistin two-

fold dilutions of 0.25-16 mg/L designed to test simultaneously four isolates. The result
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can be read visually or automatically. A recent study conducted by EUCAST, which

assessed the SensiTest colistin in comparison with BMD on 22 isolates of
Acinetobacter spp., found EA 71% and CA 82%, with 4 out of 14 susceptible isolates
recorded as false-resistant, but no false-susceptible results [35]. In contrast, in another
study that included only 10 Acinetobacter spp., the test performance appeared
excellent, with EA 100% and no false-resistant or false-susceptible results [41].
Thereby, new studies using different collections of isolates are needed to provide
further insights on the validity of SensiTest for colistin ST.
 vi) MICRONAUT-S. The MICRONAUT-S (MERLIN Diagnostika Gmbh) is composed
of a 96-well microplate offered in standard format of antimicrobials or custom format
for automated or manual ST. A single study has evaluated the MICRONAUT-S in
comparison to BMD for colistin on 22 Acinetobacter spp., and reported EA of 91%
and CA of 86%, with no false-susceptible results, whilst 3 out of 14 colistin-
susceptible isolates were found false-resistant [35].

vii) MICRONAUT MIC-Strip. The MICRONAUT MIC-Strip (MERLIN Diagnostika

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Gmbh) is a 12-well panel with colistin concentrations ranging from 0.0625 to 64 mg/L,

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designed to test one isolate. The only available study in the literature reporting on the

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performance of this product in Acinetobacter spp. showed that MICRONAUT MIC-
Strip achieved an EA of 100% and a CA of 86% with BMD, categorizing 19 out of 22

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isolates correctly [35]. The method yielded no false-susceptible results, while 3 out of
14 colistin-susceptible isolates were found false-resistant.
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viii) UMIC. The UMIC (Biocentric) is also a single-isolate test containing colistin
concentrations of 0.0625-64 mg/L. In the study performed by EUCAST on 22
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Acinetobacter spp. isolates, UMIC gained EA and CA rates of 77% and 91%,
respectively, compared with BMD, with 2 out of 8 isolates categorized incorrectly as
susceptible and without false-resistant results (35). However, another study compared
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UMIC with the reference BMD found that it produced CA of 100% using 12 A.
baumanii isolates [39].
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ix) Accelerate Pheno system. The Accelerate Pheno system (Accelerate Diagnostics) is
a fully automated system that provides antimicrobial ST results directly from positive
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blood cultures in approximately 7 h, namely more rapidly than any other commercial
automated system [43]. The MIC determination and interpretation of results is based
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on morphokinetic cellular analysis by automated microscopy of dividing individual

living cells grown in micro-colonies while being exposed to antibiotics. This system
received a de novo classification from the FDA because the technology is the only
phenotypic antimicrobial ST system applied directly from positive blood cultures. The
accuracy of the Accelerate Pheno system for colistin was tested on 136 A. baumannii
samples (132 colistin-susceptible, 4 colistin-resistant) and 90.4% EA and 91.9% CA
were observed in comparison to BMD, with 1 false-susceptible and 10 false-resistant
results [43].
 2.1.4. Interpretative criteria and internal quality control

The recommended colistin MIC breakpoints for Acinetobacter spp. are virtually the
same for CLSI and EUCAST (S≤2/R≥4 mg/L and S≤2/R>2 mg/L, respectively). As far
as polymyxin B, only CLSI (S≤2/R≥4 mg/L) and not EUCAST provide interpretative
criteria for Acinetobacter spp. [59, 60].
According to CLSI and EUCAST guidelines for polymyxins, quality control

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(QC) must be performed using the susceptible reference strains E. coli ATCC 25922

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and P. aeruginosa ATCC 27853 [59, 60]. However, most discrepancies between
reference method and gradient tests were found for BMD MICs of 2, 4 and 8 mg/L,

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while the MICs of the QC strains are lower. For this reason, EUCAST recently
recommended the addition of the colistin-resistant strain E. coli NCTC 13846 (mcr-1

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positive) in QC, the colistin MIC target value of which is 4 mg/L and occasionally
should be 2 or 8 mg/L [16, 60].
an
2.1.5. Agreement between colistin and polymyxin B
M
The CLSI in its latest document on the performance standards for antimicrobial ST
added a comment regarding the use of colistin as a surrogate for testing and reporting
polymyxin B for A. baumannii complex (i.e. colistin MICs predict polymyxin B MICs)
ed

[59]. Limited data are available regarding the correlation between colistin and
polymyxin B MICs. A study including 1,068 Acinetobacter spp. reported that colistin
pt

and polymyxin B MIC values produced by the Sensititre were within ±1 doubling
dilution for >99% of isolates, while CA between colistin and polymyxin B was 98.9%
ce

[63]. Similarly, another study that comprised 42 CRAB isolates showed a high level of
agreement between colistin and polymyxin B MICs, determined by BMD using glass-
Ac

coated or polystyrene plates (EA and CA of 100%). However, the respective AD MICs
produced EA and CA of 76.2% and 85.7% respectively, with the modes being within
±1 doubling dilution and the geometrical mean of colistin being significantly higher
than that of polymyxin B. The Etest had EA of 33.3% and CA of 100%, with the mode
and geometrical mean of colistin being significantly lower than polymyxin B [40].
2.2. Novel phenotypic assays for detection of polymyxin esistance
The polymyxin resistance can be detected in the clinical microbiological laboratory by
a number of novel phenotypic assays, including selective agar media, qualitative
 assays, and Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass
Spectrometry (MALDI-TOF MS).

2.2.1. Selective agar-based bacterial culture media

Selective culture media have recently been developed for detection of polymyxin-
resistant Gram-negative microorganisms from pure cultures and stool samples. These
media contain relatively low concentrations of colistin (3.5 or 4 mg/L) allowing the

t
inhibition of susceptible and growth of polymyxin-resistant isolates and also,

ip
daptomycin/oxazolidinones/vancomycin and amphotericin B to inhibit Gram-positive
bacteria and fungi, respectively [64–66]. They give results in 24-48 hours and could be

cr
easily used as screening tools for colistin resistance in routine clinical microbiological
laboratories. However, further studies for assessment of their performance are

us
necessitated, especially in larger collections of non-fermenters, including A.
baumannii.
an
i) SuperPolymyxinTM. The colistin-containing SuperPolymyxinTM selective culture
medium was developed for screening of any type of polymyxin-resistant Gram-
M

negative microorganism, regardless of its resistance mechanism and of its level [64].
Currently, the medium is commercially available by Elitech Microbiology
ed

(www.elitechgroup.com/france) or can be prepared in-house as described by

Nordmann et al [64]. It is based on the eosin methylene blue (EMB) medium, which is
selective for Gram-negative bacteria and contributes to species identification by
pt

differentiating lactose fermenters from non-fermenters. The method is useful for

detection of carriers and possible outbreaks due to polymyxin-resistant bacteria, thus
ce

allowing the prevention of their dissemination. When compared to BMD it exhibited

100% sensitivity and specificity for detection of polymyxin-susceptible or -resistant
Ac

isolates, including A. baumannii [64].

ii) CHROMagar COL-APSE. CHROMagar COL-APSE is another sensitive and

specific chromogenic culture medium for the isolation and identification of polymyxin-
resistant Gram-negative bacteria. This medium was found to be similar to
SuperPolymyxin for the selective growth of polymyxin-resistant organisms, but it also
provides the advantage of presumptive chromogenic identification [65].

iii) LBJMR. Another screening tool for colistin-resistant isolates from clinical samples,
 which has recently been patented is the LBJMR (Lucie Bardet-Jean-Marc Rolain)
medium [66]. In fact, it is a polyvalent selective culture medium, which can isolate
both colistin-resistant Gram-negative bacteria and vancomycin-resistant Gram-positive
bacteria, regardless of their resistance level or mechanism. The LBJMR medium was
tested on fecal samples and pure cultures and showed excellent performance on
detecting polymyxin-susceptible or -resistant isolates, including Acinetobacter.

2.2.2. Qualitative assays for detection of colistin resistance

t
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i) Rapid Polymyxin™ Acinetobacter assay. Recently the Rapid Polymyxin

cr
Acinetobacter test has been developed by Elitech Microbiology
(www.elitechgroup.com/france), in order to detect polymyxin-resistant and polymyxin-

us
susceptible A. baumannii isolates [67]. It is a liquid method based on the colorimetric
detection of a rapid metabolism related to bacterial growth, in the presence of a defined
concentration of colistin. Bacterial growth induces acid formation, which is visible by
an
the color change of the red phenol used as pH indicator (red color shifts to yellow or
orange). The method is easy-to-use and the most important feature is that it offers a
M
turn-around time to result between 3 and 4 hours, thus allowing the detection of
polymyxin resistance from bacterial cultures much earlier than any other method
currently available for Acinetobacter and therefore timely administration of the
ed

appropriate treatment and optimization of antibiotic stewardship [67]. Compared with

BMD, the Rapid Polymyxin Acinetobacter test identified correctly the 9 colistin-
pt

susceptible and the 12 colistin-resistant A. baumannii isolates tested, without false-

resistant and false-susceptible results (specificity and sensitivity 100%) [67]. However,
ce

future studies for evaluation of its performance with a greater number of isolates and
its applicability directly from clinical specimens are needed.
Ac

ii) Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test. The Rapid

ResaPolymyxin Acinetobacter/Pseudomonas NP test is a cheap and convenient assay
that rapidly (less than 4 hours) detects polymyxin resistance in non-fermenting bacteria
[68]. The test is based on detection of viable cells after growth in a medium containing
a defined concentration of colistin, which is visualized after the addition of the colorant
resazurin by its color change (blue to purple or pink). The method was applied on 92
colistin-resistant and colistin-susceptible A. baumannii and Pseudomonas aeruginosa
 isolates and showed sensitivity and specificity of 100% and 95%, respectively,
compared with the standard BMD method. These data suggested that it could be useful
in regions where colistin-resistant non-fermenters have become endemic [68].

iii) Micromax Assay. The Micromax assay (Halotech DNA SL, Madrid, Spain) is a
rapid, simple, and reliable method for determining resistance to a relevant antibiotic
such as colistin in clinical A. baumannii isolates [69]. The principle of the method is
based on the determination by fluorescence microscopy of DNA fragmentation and cell

t
wall damage, which have shown to be excellent parameters for discriminating

ip
susceptible and resistant isolates. Resistance corresponds to ≤11% of bacteria with cell

cr
wall damage after incubation with 0.5 mg/L colistin. The method was tested on a
collection of 70 A. baumannii isolates (50 colistin-susceptible, 20 colistin-resistant)

us
and identified resistant A. baumannii with 100% sensitivity and 96% specificity
according to CLSI standards. The methodology is manual or semiautomatic and could
yield results in 3 h 30 min, i.e. much sooner than MIC based polymyxin ST methods.
an
However, it presents the disadvantage of requiring specific equipment, such as
epifluorescence microscope and cell scoring.
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2.2.3. MALDI-TOF MS
ed

The MALDI-TOF MS technology is implemented in many clinical microbiological

laboratories for the identification of bacteria and fungi. Encouraging preliminary data
indicated that the technique can be applied with low cost for the rapid detection of
pt

colistin resistance [70, 71]. It has been previously shown that colistin-resistant A.
baumannii are characterized by modifications of the lipid A that can be visible by
ce

MALDI-TOF mass spectrometry [72, 73]. Relying on this observation, a large cohort
study of 284 single-patient Acinetobacter spp. clinical isolates was tested recently to
Ac

evaluate the correlation between the colistin MIC resistance profiles produced by BMD
and AD assays with the lipid A modification profiles determined by mass spectrometry
analysis of extracted membrane glycolipids. Sensitivity of 92.9% and specificity of
94% was interestingly shown [71]. Colistin-resistant isolates were defined by the
presence of an additional signature ion at m/z 2033, representing the addition of
phosphoethanolamine to one of the phosphate moieties of the m/z 1910 structure. The
authors concluded that the glycolipid mass spectrometry profiling is able to detect
colistin resistance in A. baumannii and could be an effective tool to optimize
 antimicrobial stewardship. Moreover, great effort is being currently exerted to design a
more applicable extraction protocol, which minimizes the turnaround time of the
MALDI-TOF procedure. The MALDIxin test is a MALDI-TOF-based assay, which is
able to detect within less than 15 minutes lipid A modifications associated with colistin
resistance directly on intact bacteria, with a very limited sample preparation prior to
MALDI-TOF analysis [70]. The assay was evaluated on a collection of 17 A.
baumannii isolates including 9 colistin-resistant and 8 colistin-susceptible isolates and
resulted in accurate detection of all colistin-resistant isolates. In particular, the authors

t
ip
observed that all colistin-resistant isolates were characterized by the appearance of two
peaks corresponding to the phosphoethanolamine modified lipid A (m/z 1935.3 and m/z

cr
2033.3), which were absent from colistin-susceptible isolates. This test seems to be
very promising, but further optimizations and assessment on a large scale of isolates

us
are required in the future [70].
an
3. Conclusions
Τhe application of polymyxin ST is now essential in the clinical microbiological
labοratory because of the increasing prevalence of CRAB and hence the growing need
M

for polymyxin administration. Although progress has recently been made in

standardizing polymyxin ST methods, the optimal method for polymyxin ST in routine
ed

laboratories is still challenging. Diffusion methods should not be used, while the semi-
automated systems seem to have poor performance. Novel BMD-based products are
now accessible, but evidence on their suitability in daily practice is limited. On the
pt

other hand, methods capable of detecting polymyxin-resistant A. baumannii are

constantly developing, focusing on reducing the total time required to obtain the
ce

results. The emerging MALDI-TOF technology may offer a useful tool in the future
for the detection of polymyxin-resistant A. baumannii. The variety of methods
Ac

currently available for polymyxin ST strongly emphasizes the need for harmonized
international guidelines.

4. Expert opinion
The rapid and accurate detection of polymyxin-resistant A. baumannii is of key
importance, since polymyxins often need to be used, alone or combined with other
antimicrobials as last-line drugs against A. baumannii infections, especially in regions
with high prevalence of CRAB, such as Greece, Italy, Taiwan and China. Thus far,
 several studies have evaluated the performance of polymyxin ST methods, displaying
controversial results. The ISO-standard BMD method is according to the CLSI/
EUCAST recommendations the gold standard method yielding accurate polymyxin ST
results, however it is labor-intensive and time-consuming and thus not convenient for
implementation in routine clinical microbiological laboratory.
Τhe use of gradient diffusion tests has to be avoided, due to excessive rates of
false-susceptible results. However, laboratories with limited diagnostic tools still
employ the gradient diffusion tests for polymyxin ST. Ιn these cases the results should

t
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be confirmed by BMD when the gradient diffusion MICs are near the sensitive
breakpoints (MIC of 1-2 mg/L) and when polymyxin is going to be used for the

cr
treatment. Due to the discrepancies in interpretation of results with gradient diffusion,
the usage of automated systems for polymyxin MIC determination is constantly

us
increased. Although the automated systems are generally more reliable in relation to
diffusion methods, concerns about their accuracy also seem to exist. It has been shown
an
that Phoenix100 and Vitek2 significantly underestimate colistin-resistant A.
baumannii, potentially leading to inappropriate colistin administration, while
MicroScan also seems to be unsuitable for colistin ST of Acinetobacter species. For
M

these reasons, a number of new commercial BMD products, such as SensiTest,

MICRONAUT-S, MICRONAUT MIC-Strip and UMIC have been launched and could
ed

be useful tools for polymyxin ST, but they have not yet been sufficiently assessed.
Ιn recent years, a great effort has been made in discovering new technologies,
which allow faster detection of polymyxin-resistant A. baumannii. These assays
pt

include the usage of selective culture media, qualitative assays and MALDI-TOF MS,
ce

however, more research is required to prove their reliability in the routine practice.
The selective agar-based bacterial culture media could be easily used as screening tools
for colistin resistance in routine clinical microbiological laboratories. The qualitative
Ac

techniques Rapid Polymyxin™ Acinetobacter assay and Rapid ResaPolymyxin

Acinetobacter/Pseudomonas NP test are easy-to-use methods for detection of
polymyxin resistance from bacterial cultures and offer results in 3-4 hours, allowing
the timely administration of the proper drug and thus preventing the emergence of
antibiotic resistance. Moreover, the MALDI-TOF technology could be a good and
cost-effective choice in the future for the rapid and accurate detection of polymyxin-
resistant A. baumannii, by directly assessing modification of lipid A in intact bacteria.
We should note that apart from rapid and valid determination of colistin MIC
 there are many other key issues that need attention to avoid treatment failure with
colistin against Acinetobacter, such as appropriate dosing, dosing adjustment during
continuous renal replacement therapy (CRRT) or therapeutic drug monitoring (TDM)
of colistin. The appropriate colistin dosing is still concerning, with the recommended
schemes to currently include a loading dose of 9 million units, followed by 4.5 million
units twice daily [74]. Nevertheless, in many countries, no loading dose is given and
the patients are receiving lower doses of 3 million units two times a day, which may be
responsible for failures. Furthermore, during CRRT the doses have to be increased

t
ip
[75], however in many cases no loading dose is given and the maintenance dose is not
increased, possibly leading to treatment failures. Also, TDM is crucial to adjust the

cr
colistin dose for individual patients [76, 77]. Another issue of debate is the
administration of colistin as single agent or in combinations, with increasing reports to

us
suggest that colistin monotherapy was associated with better outcomes compared to the
colistin-meropenem combination therapy [74, 78]. Finally, the
an
pharmacokinetic/pharmacodynamic (PK/PD) characteristics of colistin are not well
explored and there is still some debate about whether colistin is time-dependent or
dose-dependent or both. For instance, using in vitro PK/PD modelling of colistin
M

activity against K. pneumoniae, we recently reported that fAUC/MIC is the best

predictor of colistin activity for clinically achievable concentrations, and fCmax/MIC
ed

is the best determinant of colistin bactericidal activity [76]. This debate may have some
impact when analyzing the MICs, pointing out the possible need to revise current
susceptibility breakpoints [76]. Altogether, the above issues need to be considered at
pt

the same time of the MIC determination, to avoid failures.

ce

In conclusion, the optimal method for assessing polymyxin resistance in routine

microbiological laboratories is still challenging. Due to intensive research, it can be
anticipated that within five years much progress will be made towards the optimization
Ac

and standardization of available polymyxin ST, to facilitate the accurate detection of

polymyxin-resistant A. baumannii. Also, the efforts to discover new methods with the
main goal of minimizing the time obtaining results are expected to be intensified.
 Funding
This paper was not funded.

Declaration of interest
The authors have no relevant affiliations or financial involvement with any
organization or entity with a financial interest in or financial conflict with the subject
matter or materials discussed in the manuscript. This includes employment,
consultancies, honoraria, stock ownership or options, expert testimony, grants or

t
ip
patents received or pending, or royalties.

cr
Reviewer disclosures
Peer reviewers on this manuscript have no relevant financial or other relationships to

us
disclose.
an
M
ed
pt
ce
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 References
Papers of special note have been highlighted as:
* of interest
** of considerable interest.

1. Dijkshoorn L, Nemec A, Seifert H. An increasing threat in hospitals: multidrug-

resistant Acinetobacter baumannii. Nat Rev Microbiol.2007;5(12):939-51.
2. Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a

t
ip
successful pathogen. Clin Microbiol Rev. 2008;21(3):538-82.
3. Visca P, Seifert H, Towner KJ. Acinetobacter infection-an emerging threat to human

cr
health. IUBMB Life. 2011;63(12):1048-54.
4. Isler B, Doi Y, Bonomo RA, Paterson DL. New treatment options against

us
carbapenem-resistant Acinetobacter baumannii infections. Antimicrob Agents
Chemother. 2018;63(1):e01110-18.
5.
an
Zarrilli R, Pournaras S, Giannouli M, Tsakris A. Global evolution of multidrug-
resistant Acinetobacter baumannii clonal lineages. Int J Antimicrob Agents.
2013;41(1):11-9.
M

6. Tacconelli E, Carrara E, Savoldi A, et al. Discovery, research, and development of

new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis.
ed

Lancet Infect Dis. 2018; 18(3):318-27.

7. Durante-Mangoni E, Zarrilli R. Global spread of drug-resistant Acinetobacter
baumannii: molecular epidemiology and management of antimicrobial resistance.
pt

Future Microbiol. 2011;6(4):407-22.

8. Pogue JM, Mann T, Barber KE, Kaye KS. Carbapenem-resistant Acinetobacter
ce

baumannii: epidemiology, surveillance and management. Expert Rev Anti Infect

Ther. 2013;11(4):383-93.
Ac

9. Li J, Nation RL, Milne RW, Turnidge JD, Coulthard K. Evaluation of colistin as an

agent against multi-resistant Gram-negative bacteria. Int J Antimicrob Agents.
2005;25(1):11-25.
10. Cai Y, Chai D, Wang R, Liang B, Bai N. Colistin resistance of Acinetobacter
baumannii: clinical reports, mechanisms and antimicrobial strategies. J Antimicrob
Chemother. 2012;67(7):1607-15.
11. Jeannot K, Bolard A, Plésiat P. Resistance to polymyxins in Gram-negative
organisms. Int J Antimicrob Agents. 2017;49(5):526-35.
 12. Nowak J, Zander E, Stefanik D, et al. High incidence of pandrug-resistant
Acinetobacter baumannii isolates collected from patients with ventilator-associated
pneumonia in Greece, Italy and Spain as part of the MagicBullet clinical trial. J
Antimicrob Chemother. 2017;72(12):3277-82.
13. Humphries RM. Susceptibility testing of the polymyxins: where are we now?
Pharmacotherapy. 2015;35(1):22-7.
** Comprehensive review on susceptibility testing methods of the polymyxins.
14. Vasoo S. Susceptibility Testing for the Polymyxins: Two Steps Back, Three Steps

t
ip
Forward? J Clin Microbiol. 2017;55(9):2573-82.
15. EUCAST. Recommendations for Colistin (Polymyxin E) MIC Testing-Joint EUCAST

cr
and CLSI Recommendation. 2016. Available from:
http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/General_documents/

us
Recommendations_for_MIC_determination_of_colistin_March_2016.pdf. [accessed
06.09.2019].
an
** Gives the recommendations for MIC determination of colistin as recommended by
the joint CLSI-EUCAST Polymyxin Breakpoints Working Group.
16. EUCAST. EUCAST Warnings Concerning Antimicrobial Susceptibility Testing
M

Products or Procedures-Antimicrobial Susceptibility Testing of Colistin-Problems

Detected with Several Commercially Available Products. 2016. Available from:
ed

http://www.eucast.org/ast_of_bacteria/warnings/#c13111. [accessed 06.09.2019].

** Describes the problems with colistin susceptibility testing and several
commercially available products.
pt

17. Gales AC, Reis AO, Jones RN. Contemporary assessment of antimicrobial
susceptibility testing methods for polymyxin B and colistin: review of available
ce

interpretative criteria and quality control guidelines. J Clin Microbiol. 2001;39(1):

183-190.
Ac

18. Reis AO, Luz DA, Tognim MC, Sader HS, Gales AC. Polymyxin-resistant
Acinetobacter spp. isolates: what is next? Emerg Infect Dis. 2003;9(8):1025-7.
19. Arroyo LA, García-Curiel A, Pachón-Ibañez ME, et al. Reliability of the E-test method
for detection of colistin resistance in clinical isolates of Acinetobacter baumannii. J
Clin Microbiol. 2005;43(2):903-5.
20. Tan TY, Ng LS. Comparison of three standardized disc susceptibility testing
methods for colistin. J Antimicrob Chemother. 2006;58(4):864-7.
21. Goldstein FW, Ly A, Kitzis MD. Comparison of Etest with agar dilution for
 testing the susceptibility of Pseudomonas aeruginosa and other multidrug-resistant
bacteria to colistin. J Antimicrob Chemother. 2007;59(5):1039-40.
22. Lo-Ten-Foe JR, de Smet AM, Diederen BM, Kluytmans JA, van Keulen PH.
Comparative evaluation of the VITEK 2, disk diffusion, etest, broth microdilution,
and agar dilution susceptibility testing methods for colistin in clinical isolates,
including heteroresistant Enterobacter cloacae and Acinetobacter baumannii
strains. Antimicrob Agents Chemother. 2007;51(10):3726-30.
23. Tan TY, Ng SY. Comparison of Etest, Vitek and agar dilution for susceptibility

t
ip
testing of colistin. Clin Microbiol Infect. 2007;13(5):541-4.
24. Tan TY, Ng LS, Poh K. Susceptibility testing of unconventional antibiotics against

cr
multiresistant Acinetobacter spp. by agar dilution and Vitek 2. Diagn Microbiol
Infect Dis. 2007;58(3):357-61.

us
25. Behera B, Mathur P, Das A, et al. Evaluation of susceptibility testing methods for
polymyxin. Int J Infect Dis. 2010;14(7):e596-601.
an
26. Nemec A, Dijkshoorn L. Variations in colistin susceptibility among different
species of the genus Acinetobacter. J Antimicrob Chemother. 2009;65(2):367-9.
27. Girardello R, Bispo PJ, Yamanaka TM, Gales AC. Cation concentration variability
M

of four distinct Mueller-Hinton agar brands influences polymyxin B susceptibility

results. J Clin Microbiol. 2012;50(7):2414-8.
ed

28. Lee SY, Shin JH, Lee K, et al. Comparison of the Vitek 2, MicroScan, and Etest
methods with the agar dilution method in assessing colistin susceptibility of
bloodstream isolates of Acinetobacter species from a Korean university hospital. J
pt

Clin Microbiol. 2013;51(6):1924-6.

ce

29. Hindler JA, Humphries RM. Colistin MIC variability by method for contemporary
clinical isolates of multidrug-resistant gram-negative bacilli. J Clin Microbiol.
2013;51(6):1678-84.
Ac

* Reports poor performance of the Etest with three different commercial

manufacturers of prepared MHA.
30. Albur M, Noel A, Bowker K, Macgowan A. Colistin susceptibility testing: time
for a review. J Antimicrob Chemother. 2014;69(5):1432-4.
31. Piewngam P, Kiratisin P. Comparative assessment of antimicrobial susceptibility
testing for tigecycline and colistin against Acinetobacter baumannii clinical
isolates, including multidrug-resistant isolates. Int J Antimicrob Agents.
2014;44(5):396–401.
 32. Nhung PH, Miyoshi-Akiyama T, Phuong DM, et al. Evaluation of the Etest
method for detecting colistin susceptibility of multidrug-resistant Gram-negative
isolates in Vietnam. J Infect Chemother. 2015;21(8):617-9.
33. Dafopoulou K, Zarkotou O, Dimitroulia E, et al. Comparative evaluation of
colistin susceptibility testing methods among carbapenem-nonsusceptible
Klebsiella pneumoniae and Acinetobacter baumannii clinical isolates. Antimicrob
Agents Chemother. 2015;59(8):4625–30.
34. Vourli S, Dafopoulou K, Vrioni G, Tsakris A, Pournaras S. Evaluation of two

t
ip
automated systems for colistin susceptibility testing of carbapenem-resistant
Acinetobacter baumannii clinical isolates. J Antimicrob Chemother.

cr
2017;72(9):2528-30.
35. Matuschek E, Åhman J, Webster C, Kahlmeter G. Antimicrobial susceptibility

us
testing of colistin - evaluation of seven commercial MIC products against standard
broth microdilution for Escherichia coli, Klebsiella pneumoniae, Pseudomonas
an
aeruginosa, and Acinetobacter spp. Clin Microbiol Infect. 2018;24(8):865-70.
* Assessment of five commercial BMD products and two gradient tests for colistin
MIC determination.
M

36. Li H, Zhou H, Li X, et al. The evaluation of four in vitro susceptibility testing

methods for colistin on carbapenem-resistant Acinetobacter baumannii.
ed

Jundishapur J Microbiol. 2017;10(9):e55956.

37. Simar S, Sibley D, Ashcraft D, Pankey G. Colistin and polymyxin B minimal
inhibitory concentrations determined by Etest found unreliable for Gram-negative
pt

bacilli. Ochsner J. 2017;17(3):239-42.

ce

38. Bakthavatchalam YD, Veeraraghavan B, Shankar A, Thukaram B, Krishnan DN.

Evaluation of colistin and polymyxin B susceptibility testing methods in
Klebsiella pneumoniae and Acinetobacter baumannii. J Infect Dev Ctries.
Ac

2018;12(6):504-7.
39. Jayol A, Nordmann P, André C, Poirel L, Dubois V. Evaluation of three broth
microdilution systems to determine colistin susceptibility of Gram-negative bacilli.
J Antimicrob Chemother. 2018;73(5):1272-8.
40. Singhal L, Sharma M, Verma S, et al. Comparative evaluation of broth
microdilution with polystyrene and glass-coated plates, agar dilution, E-test, Vitek,
and disk diffusion for susceptibility testing of colistin and polymyxin B on
carbapenem-resistant clinical isolates of Acinetobacter baumannii. Microb Drug
 Resist. 2018;24(8):1082-8.
41. Carretto E, Brovarone F, Russello G, et al. Clinical validation of SensiTest
Colistin, a broth microdilution-based method to evaluate colistin MICs. J Clin
Microbiol. 2018;56(4):e01523-17.
42. Lellouche J, Schwartz D, Elmalech N, et al. Combining VITEK® 2 with colistin
agar dilution screening assist timely reporting of colistin susceptibility. Clin
Microbiol Infect. 2019; 25(6):711-6.
43. Pancholi P, Carroll KC, Buchan BW, et al. Multicenter evaluation of the

t
ip
Accelerate PhenoTest BC kit for rapid identification and phenotypic antimicrobial
susceptibility testing using morphokinetic cellular analysis. J Clin Microbiol.

cr
2018;56(4):e01329-17.
44. Simner PJ, Bergman Y, Trejo M, et al. Two-site evaluation of the colistin broth

us
disk elution test to determine colistin in vitro activity against Gram-Negative
bacilli. J Clin Microbiol. 2019;57(2):e01163-18.
an
45. International Organization for Standardization (ISO). Clinical Laboratory Testing
and In Vitro Diagnostic Test Systems. Susceptibility Testing of Infectious Agents
and Evaluation of Performance of Antimicrobial Susceptibility Test Devices. Part
M

2: Evaluation of Performance of Antimicrobial Susceptibility Test Devices.

International Standard 20776-2. International Organization for Standardization;
ed

Geneva, Switzerland: 2007.

46. International Organization for Standardization (ISO). Clinical Laboratory Testing
and In Vitro Diagnostic Test Systems. Susceptibility Testing of Infectious Agents
pt

and Evaluation of Performance of Antimicrobial Susceptibility Test Devices. Part

ce

1: Reference Method for Testing the In Vitro Activity of Antimicrobial Agents

against Rapidly Growing Aerobic Bacteria Involved in Infectious Diseases.
47. Hawley JS, Murray CK, Griffith ME, et al. Susceptibility of acinetobacter strains
Ac

isolated from deployed U.S. military personnel. Antimicrob Agents Chemother.

2007;51(1):376-78.
48. Karvanen M, Malmberg C, Lagerbäck P, Friberg LE, Cars O. Colistin is
extensively lost during standard in vitro experimental conditions. Antimicrob
Agents Chemother. 2017; 61(11):e00857-17.
49. Nation RL, Li J, Cars O, et al. Framework for optimisation of the clinical use of
colistin and polymyxin B: the Prato polymyxin consensus. Lancet Infect Dis.
2015;15(2):225-34.
 50. Sader HS, Rhomberg PR, Flamm RK, Jones RN. Use of a surfactant (polysorbate
80) to improve MIC susceptibility testing results for polymyxin B and colistin.
Diagn Microbiol Infect Dis. 2012;74(4):412-4.
51. Kavanagh A, Ramu S, Gong Y, Cooper MA, Blaskovich MAT. Effects of
microplate type and broth additives on microdilution MIC susceptibility assays.
Antimicrob Agents Chemother. 2018;63(1):e01760-18.
52. Turnidge JD. Susceptibility testing issues with old antibiotics. Presented at:
ESCMID Conference on Reviving Old Antibiotics; 2014 October 22-24; Vienna,

t
ip
Austria.
53. Tam VH, Cao H, Ledesma KR, Hu M. In vitro potency of various polymyxin B

cr
components. Antimicrob Agents Chemother. 2011;55(9):4490-1.
54. Kassamali Z, Prince RA, Danziger LH, Rotschafer JC, Rhomberg PR, Jones RN.

us
Microbiological assessment of polymyxin B components tested alone and in
combination. Antimicrob Agents Chemother. 2015;59(12):7823-5.
an
55. Diep JK, Covelli J, Sharma R, et al. Comparison of the composition and in vitro
activity of polymyxin B products. Int J Antimicrob Agents. 2018; 52(3):365-71.
56. Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial
M

Susceptibility Tests for Bacteria That Grow Aerobically. Eleventh edition. CLSI
standard M07-A11. Clinical and Laboratory Standards Institute; Wayne, PA,
ed

USA: 2018.
57. Matzneller P, Strommer S, Österreicher Z, Mitteregger D, Zeitlinger M. Target
site antimicrobial activity of colistin might be misestimated if tested in
pt

conventional growth media. Eur J Clin Microbiol Infect Dis. 2015;34(10):1989-

ce

94.
58. Turlej-Rogacka A, Xavier BB, Janssens L, et al. Evaluation of colistin stability in
agar and comparison of four methods for MIC testing of colistin. Eur J Clin
Ac

Microbiol Infect Dis. 2018;37(2):345-53.

59. Clinical and Laboratory Standards Institute. Performance Standards for
Antimicrobial Susceptibility Testing; Twenty-Ninth Informational Supplement.
CLSI document M100-S29. Clinical and Laboratory Standards Institute; Wayne,
PA, USA: 2019.
60. EUCAST. Breakpoint Tables for Interpretation of MICs and Zone Diameters.
Version 9.0. 2019. Available from: http://www.eucast.org/clinical_breakpoints/.
[accessed 06.09.2019].
 61. Dafopoulou K, Zarkotou O, Dimitroulia E, et al. Reply to “Reliability of gradient
diffusion methods for detection of acquired colistin resistance”. Antimicrob
Agents Chemother. 2016;60(7):4424-5.
62. U.S. Food and Drug Administration. Z-2770-2017-Class 2 device recall VITEK 2
Gram negative test kit containing colistin (cs01n). FDA; Silver Spring, MD: 2017.
63. Sader HS, Rhomberg PR, Farrell DJ, Jones RN. Differences in potency and
categorical agreement between colistin and polymyxin B when testing 15,377
clinical strains collected worldwide. Diagn Microbiol Infect Dis. 2015;83(4):379-

t
ip
81.
64. Nordmann P, Jayol A, Poirel L. A universal culture medium for screening

cr
polymyxin-resistant gram-negative isolates. J Clin Microbiol. 2016;54(5):1395-9.
65. Abdul Momin MHF, Bean DC, Hendriksen RS, Haenni M, Phee LM, Wareham

us
DW. CHROMagar COL-APSE: a selective bacterial culture medium for the
isolation and differentiation of colistin-resistant Gram-negative pathogens. J Med
an
Microbiol. 2017;66(11):1554-61.
66. Bardet L, Le Page S, Leangapichart T, Rolain JM. LBJMR medium: a new
polyvalent culture medium for isolating and selecting vancomycin and colistin-
M

resistant bacteria. BMC Microbiol. 2017;17(1):220.

67. Lescat M, Poirel L, Jayol A, Nordmann P. Performances of the Rapid Polymyxin
ed

Acinetobacter and Pseudomonas tests for colistin susceptibility testing. Microb

Drug Resist. 2019;25(4):520-3.
68. Lescat M, Poirel L, Tinguely C, Nordmann P. A resazurin reduction-based assay
pt

for rapid detection of polymyxin resistance in Acinetobacter baumannii and

ce

Pseudomonas aeruginosa. J Clin Microbiol. 2019;57(3):e01563-18.

69. Tamayo M, Santiso R, Otero F, et al. Rapid determination of colistin resistance in
clinical strains of Acinetobacter baumannii by use of the micromax assay. J Clin
Ac

Microbiol. 2013;51(11):3675-82.
70. Dortet L, Potron A, Bonnin RA, et al. Rapid detection of colistin resistance in
Acinetobacter baumannii using MALDI-TOF-based lipidomics on intact bacteria.
Sci Rep. 2018;8(1):16910.
71. Leung LM, McElheny CL, Gardner FM, et al. A prospective study of
Acinetobacter baumannii complex isolates and colistin susceptibility monitoring
by mass spectrometry of microbial membrane glycolipids. J Clin Microbiol. 2019;
57(3):e01100-18.
 72. Beceiro A, Llobet E, Aranda J, et al. Phosphoethanolamine modification of lipid A
in colistin-resistant variants of Acinetobacter baumannii mediated by the pmrAB
two-component regulatory system. Antimicrob Agents Chemother.
2011;55(7):3370-9.
73. Pelletier MR, Casella LG, Jones JW, et al. Unique structural modifications are
present in the lipopolysaccharide from colistin-resistant strains of Acinetobacter
baumannii. Antimicrob Agents Chemother. 2013;57(10):4831-40.
74. Paul M, Daikos GL, Durante-Mangoni E, et al. Colistin alone versus colistin plus

t
ip
meropenem for treatment of severe infections caused by carbapenem-resistant
Gram-negative bacteria: an open-label, randomised controlled trial. Lancet Infect

cr
Dis. 2018;18(4):391-400.
75. Nation RL, Garonzik SM, Thamlikitkul V, et al. Dosing guidance for intravenous

us
colistin in critically-ill patients. Clin Infect Dis. 2017;64(5):565-71.
76. Tsala M, Vourli S, Georgiou PC, et al. Exploring colistin pharmacodynamics
an
against Klebsiella pneumoniae: a need to revise current susceptibility breakpoints.
J Antimicrob Chemother. 2018;73(4):953-61.
77. Akers KS, Rowan MP, Niece KL, et al. Colistin pharmacokinetics in burn patients
M

during continuous venovenous hemofiltration. Antimicrob Agents Chemother.

2015;59(1):46-52.
ed

78. Dickstein Y, Lellouche J, Ben Dalak Amar M, et al. Treatment οutcomes of

colistin- and carbapenem-resistant Acinetobacter baumannii infections: an
exploratory subgroup analysis of a randomized clinical trial. Clin Infect Dis.
pt

2019;69(5):769-76.
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Ac
Table 1. Studies evaluating the performance of various polymyxin susceptibility testing methods for Acinetobacter spp.

Drug Isolatesb Methods testedc Reference No. (%) No. of isolates with No. of isolates
testeda method of MICs of (mg/L) with false
resistant susceptibility
isolatesd
≤1 2 4 8 ≥16

PB 60 Acinetobacter - BMD (PML BMD 3 (5) NS

spp. Microbiologics)
- DD (polymyxin Β 0 (0) NA 3 (R≤8 mm, CLSI/
300 U, BD/Difco) R≤10 mm, this study)

CS - BMD (PML BMD 3 (5) 54 3 1 1 1

Microbiologics)
- DD (colistin NS NA NS
10 μg, BD)

t
ip
PB 100 Acinetobacter - AD AD 5 (5) NS
spp. (80 A. - DD 0 (0) NA 5
baumanii)
CS 115 A. baumanii - BMD BMD 22 (19.1) 93 2 6 1 13

cr
- Etest (AB Biodisk) 20 (17.4) NS 2
CS 61 Acinetobacter - AD AD 0 (0) 37 24 0 0 0
spp. - DD (10 μg, BD, 0 (0) NA 0 (R≤8 mm,

us
Package insert; package insert);
25 μg, Oxoid, BSAC; 0 (0) 0 (R≤14 mm, BSAC);
50 μg, Oxoid, SFM) 0 (0) 0 (R≤14 mm, SFM)
CS 14 A. baumanii - AD AD 6 (42.9) NS
an
- Etest (AB Biodisk) NS NS NS
CS 10 (7 A. - BMD BMD 2 (20) 7 1 0 0 2
baumannii,
PB (only 2 Acinetobacter
M
for DD) spp., 1 A. lwoffii)
- AD (MH/ NS NS NS
Isosensitest agar)
- DD (colistin 10 μg, 6 (60) NS NS
ed

Rosco)
- DD (polymyxin B 1 (10) NA NS
150 μg, Rosco)
- Etest (AB Biodisk, NS NS NS
MH/Isosensitest agar)
pt

- Vitek2 (bioMérieux, NS NS NS
AST-N038)

Isolatesb Methods testedc

ce

Drug Reference No. (%) No. of isolates with No. of isolates

testeda method of MICs of (mg/L) with false
resistant susceptibility
isolatesd
≤1 2 4 8 ≥16
Ac

CS 58 Acinetobacter - AD AD 0 (0) NS
spp.
- Etest (AB Biodisk) 1 (1.7) NS 0
- Vitek2 (AST-N032) 0 (0) NS 0

CS 44 carbapenem- - AD AD 0 (0) 33 11 0 0 0
resistant A.
calcoaceticus- - Vitek2 (AST-N032) 0 (0) NS 0
baumannii
complex
PB 91 MDR - BMD BMD 3 (3.3) NS
Acinetobacter spp. - AD 2 (2.2) NS 1
CS (only
- Etest (AB Biodisk) 2 (2.2) NS 1
for DD)
- DD (polymyxin B 1 (1.1) NA 2 (R≤11 mm,
300 U, BBL, BD) S≥12 mm, CLSI)
- DD (colistin 2 (2.2) NA 1 (R≤10 mm,
10 μg, BBL, BD) S≥11 mm, CLSI)

CS 154 Acinetobacter - AD AD 18 (11.7) 133 3 4 1 13

spp. (20 A. - Etest (AB Biodisk) 18 (11.7) 133 3 5 2 11 NS
baumannii)
PB 10 A. baumanii - BMD 2 (20) 8 0 2 0 0

- Etest (AB

t
bioMérieux)

ip
- Oxoid MHA 2 (20) 8 0 1 0 1 0
- Difco MHA 2 (20) 8 0 1 1 0 0
- Merck MHA 1 (10) 7 2 1 0 0 1

cr
- Himedia MHA 2 (20) 8 0 2 0 0 0

CS 213 Acinetobacter - AD AD 13 (6.1) 154 46 1 2 10

spp. (94 A.
- Vitek2 (AST-N132) 11 (5.2) 182 20 2 1 8 2

us
baumanii)
- Microscan 36 (16.9) NA 177 19 17 NA 2
(Siemens, Gram
negative breakpoint
combo panel type 42)
an
- Etest (bioMérieux) 15 (7) 198 0 1 2 12 0

Drug Isolatesb Methods testedc Reference No. (%) No. of isolates with No. of isolates
testeda method of MICs of (mg/L) with false
M
resistant susceptibility
isolatesd
≤1 2 4 8 ≥16
CS Phase I: - BMD-P80 BMD-P80 7 (25.9) NS
27 MDR A.
ed

baumanii - Broth/tube 8 (29.6) NS 0

macrodilution (TDS)
- Etest (bioMérieux), 3 (11.1) NS 4
BBL MHA, BD
pt

Phase II: - BMD-P80 BMD-P80 3 (27.3) NS

11 MDR A. - BMD 5 (45.5) NS 0
baumanii
ce

- AD 3 (27.3) NS 0
- Trek GNXF 3 (27.3) NS 0
Sensititre panels
(Trek Diagnostics)
- Etest (bioMérieux)
Ac

-BBL MHA 1 ( 9.1) NS 2

-Hardy MHA 1 ( 9.1) NS 3
-Remel MHA 2 (18.2) NS 2
CS 29 Acinetobacter BMD (2 different None
spp. (29×3=87 BMD trays: non-
measurements) coated V-bottom and
tissue-culture-coated
round bottom) with or
without P-80

- NMTT 6 (6.9) 81 0 6 0 0
- NMTT+P-80 0 (0) 87 0 0 0 0
- TCMTT 35 (40.2) 19 33 17 18 0
- TCMTT+P-80 16 (18.4) 37 34 14 2 0
CS 290 A. baumanii - BMD BMD 27 (9.3)g 57 206 14 0 13
(225 MDR
isolates) - DD (colistin 10 μg, 13 (4.5) NA 14 (R≤9 mm,
Oxoid) S≥12 mm)g
- Etest (bioMérieux) 12 (4.1)g 276 2 1 7 4 15g
- Vitek2 (AST-N136) 12 (4.1)g 218 60 1 0 11 16g

CS 102 A. baumanii - BMD BMD 0 (0) 102 0 0 0 0

- Etest (bioMérieux) 0 (0) 102 0 0 0 0 0

Drug Isolatesb Methods testedc Reference No. (%) No. of isolates with No. of isolates
testeda method of MICs of (mg/L) with false
resistant susceptibility
isolatesd

t
≤1 2 4 8 ≥16

ip
CS 20 carbapenem- - BMD BMD 18 (90) 0 2 8 6 4
nonsusceptible A.
baumanii - BMD-P80 12 (60) 1 7 5 4 3 6

cr
- AD 18 (90) 1 1 4 3 11 2
- Etest (bioMérieux) 11 (55) 6 3 6 4 1 7
- MIC test strip 13 (65) 0 7 9 2 2 5
(MTS; Liofilchem)

us
- Vitek2 (AST-EXN8) 20 (100) 0 0 10 5 5 0

CS 117 CRAB - BMD BMD 29 (24.8) 76 12 15 11 3

- AD 42 (35.9) 63 12 22 5 15 1
- Phoenix100
an 18 (15.4) 86 13 7 11 NA 12
(Phoenix NMIC/
ID-96 Panel, BD)
- Vitek2 (AST-XN05) 19 (16.2) 89 9 4 1 14 11
M
CS 22 Acinetobacter BMD Reference
spp. (14 A. BMD
baumanii)
- Reference frozen 8 (36.4) 11 3 0 0 8
ed

BMD panels
according to ISO
standard 20776-1
(Thermo Fisher
Scientific)
pt

- Sensititre custom 10 (45.5)g NS 0

plate (Thermo
Fisher Scientific)
ce

- MICRONAUT-S 11 (50)g NS 0
(MERLIN Diagnostika
Gmbh)

- MICRONAUT MIC- 11 (50)g NS 0

Ac

Strip (MERLIN
Diagnostika Gmbh)
- SensiTest 12 (54.5)g NS 0
(Liofilchem)
- UMIC (Biocentric) 6 (27.3)g NS 2

Drug Isolatesb Methods testedc Reference No. (%) No. of isolates with No. of isolates
testeda method of MICs of (mg/L) with false
resistant susceptibility
isolatesd
≤1 2 4 8 ≥16
Gradient tests
 - Etest (bioMérieux), 2 (9.1)g NS 6
Oxoid MH
- Etest, BBL MH 1 (4.5)g NS 7
- Etest, bioMérieux's 4 (18.2)g NS 4
ΜΗE
- MTS (Liofilchem), 4 (18.2)g NS 4
Oxoid MH
- MTS, BBL MH 1 (4.5)g NS 7

CS 202 CRAB - BMD BMD-P80 1 (0.5)g NS 0

- BMD-P80 0 (0) NS
- E-test (AB Biodisk) 0 (0) NS 0
- AD 19 (9.4)g NS 0
CS 60 A. baumanii - BMD BMD 9 (15) NS

t
- Etest (bioMérieux) 1 (2) NS 8

ip
PB - BMD BMD 5 (8) NS
- Etest (bioMérieux) 0 (0) NS 5

cr
CS 120 A. baumanii - BMD BMD 11 (9) NS

us
- Etest (bioMérieux) 7 (6) NS 4
- Vitek2 (AST-N281, 10 (32) NS 1
n=31 isolates)
an
PB - BMD BMD 11 (9) NS
- Etest (bioMérieux) 1 (1) NS 10

CS 12 A. baumanii - BMD BMD 8 (66.6) 4 0 0 0 8

M
- Sensititre 8 (66.6) 4 0 0 8 0
(ThermoFisher
Diagnostics)
- UMIC (Biocentric) 8 (66.6) 3 1 0 0 8 0
ed

- MicroScan 10 (83.3) NA 2 0 10 NA 1
(Beckman Coulter)

Drug Isolatesb Methods testedc Reference No. (%) No. of isolates with No. of isolates
testeda
pt

method of MICs of (mg/L) with false

resistant susceptibility
isolatesd
≤1 2 4 8 ≥16
ce

CS 42 CRAB - BMD-Gs; BMD BMD-Gs/ 0 (0) 11 31 0 0 0 −/3

glass-coated plate BMD-P
- BMD-P; BMD 3 (7.2) 26 13 3 0 0 0/−
polystyrene plate
Ac

- AD 6 (14.3) 18 18 6 0 0 0/3
- DD (colistin 0 (0) NA 0/3 (R≤9 mm,
10 μg, Difco) S≥12 mm, Piewngam)
- Etest (bioMérieux) 0 (0) 41 1 0 0 0 0/3
- Vitek2 (AST-N281) 0 (0) 42 0 0 0 0 0/3

PB - BMD-Gs BMD-Gs/ 0 (0) 25 17 0 0 0 −/3

BMD-P
- BMD-P 3 (7.2) 26 13 3 0 0 0/−
- AD 0 (0) 34 8 0 0 0 0/3
- DD (polymyxin B 0 (0) NA 0/3 (R≤9 mm,
150 μg, 300U/disk, S≥12 mm, Piewngam)
Hi-Media)
- E-test (bioMérieux) 1 (2.4) 25 16 0 1 0 0/3
- Vitek2 (AST-N281) 0 (0) 42 0 0 0 0 0/3
CS 10 Acinetobacter - BMD BMD 1 (10) NS
spp. (6 A.
baumanii) - SensiTest 1 (10) NS 0
(Liofilchem)
CS 199 CRAB - BMD BMD 27 (13.6) 143 29 3 2 22
- AD NS NS 3
- Vitek2 (AST-N114, NS NS 8
AST-N308,
AST N348)
- Vitek2/AD combined NS NS 3
method
CS 136 A. baumanii - BMD BMD 4 (2.9) NS
- Accelerate Pheno NS NS 1
system (Accelerate
Diagnostics)

t
CS Site 1: - BMD (Microscan) BMD 7 (58.3) NS

ip
12 A. baumanii - CBDE 7 (58.3) NS 0

Site 2: - BMD (RUO BMD 0 (0) NS

cr
12 A. baumanii Sensititre GNX2F
panels, Thermo
Fisher)
- CBDE 0 (0) NS 0

us
Abbreviations:

a
PB, Polymyxin B; CS, colistin
b
MDR, multidrug-resistant A. baumannii; CRAB, carbapenem-resistant A. baumannii
c
BMD, broth microdilution; DD, disk diffusion; AD, agar dilution; MH, Muller-Hinton; MHA, Muller-Hinton agar;
an
BMD-P80, BMD with polysorbate 80; CBDE, colistin broth disk elution
d
For MIC methods, colistin and polymyxin B resistance was defined according to EUCAST and/or CLSI
breakpoints (S≤2/R≥4 mg/L and S≤2/R>2 mg/L, respectively) for Acinetobacter spp.
e
CA, categorical agreement
f
EA, essential agreement
M
g
Errors and CA were calculated by authors using data provided by the respective studies
h
No. (%) of resistant isolates, errors and CA were recalculated using resistance breakpoint ≥4 mg/L
NA, not applicable; NS, not specified
ed
pt
ce
Ac