International Journal of Antimicrobial Agents 54 (2019) 587–591

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International Journal of Antimicrobial Agents

journal homepage: www.elsevier.com/locate/ijantimicag

Discrepancies in susceptibility testing to colistin in Acinetobacter

baumannii: The influence of slow growth and heteroresistance
Carlos Hernán Rodriguez a,∗, German Traglia a, Nadya Bastias b, Cecilia Pandolfo b,
Geni Bruni b, Marcela Nastro a, Ruben Barrios c, Eleonora M. Bavastro d, Mariana Carol Rey e,
Isabel A. Marques e, Facundo Heger a, Carlos Vay a, Jennifer S. Fernandez f,
María Soledad Ramirez f, Angela Famiglietti a
a
Laboratorio de Bacteriología, Departamento de Bioquímica Clínica, Hospital de Clínicas José de San Martín, Facultad de Farmacia y Bioquímica,
Universidad de Buenos Aires, Buenos Aires, INFIBIOC, UBA, Argentina
b
Laboratorio de Bacteriología, Hospital del Carmen Mendoza, Argentina
c
BD, Life Sciencies, Argentina
d
Laboratorio de Bacteriología, Hospital Público Descentralizado ‘Dr. Guillermo Rawson’, San Juan, Argentina
e
Hospital ‘Dr. Julio C. Perrando’, Resistencia-Chaco, Argentina
f
Department of Biological Science, California State University Fullerton, Fullerton, CA, USA

a r t i c l e i n f o a b s t r a c t

Article history: The increasing use of polymyxins as last-resort drugs for managing infections by Acinetobacter bauman-
Received 7 May 2019 nii has led to the emergence of resistance. This study aimed to determine the resistance mechanisms in
Accepted 1 August 2019
Acinetobacter baumannii isolates with colistin MIC ≥ 4 mg/L and to relate the mechanisms of resistance
with the difficulties in detecting them. Absolute agreement among the different methodologies (Phoenix
Editor: Jean-Marc Rolain automatized system, broth and agar dilution, and a rapid colorimetric test) in the 140 colistin-susceptible
isolates was observed; whereas in the 25 resistant isolates, the performance varied according to the col-
Keywords:
istin MIC value. Most of the discrepancies (irrespective of the methodology that was used) were observed
Acinetobacter baumannii
Colistin resistance in isolates with an MIC value close to the breakpoint. The number of errors in each method in the resis-
Susceptibility testing tant isolates was as follows: rapid test, four of 25 (16%); agar dilution, eight of 25 (32%); Phoenix system,
Rapid diagnostic test 13 of 25 (52%) and its manual reading at 24 h, eight of 25 (32%). Categorical errors were detected in 13
pmrB gene isolates: slow growth was the main reason in five isolates, whereas in the remaining eight isolates, slow
lpx gene growth was detected together with a low proportion of colistin-resistant subpopulations and the colistin
MIC value was close to the breakpoint value. To understand the probable reason for the observed MIC
values, sequencing of genes associated with colistin resistance was performed. Mutations at lpxA, lpxC,
and pmrB genes were detected and it was observed that isolates carrying mutations in lpxC presented
slow growth at killing curves.
© 2019 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

1. Introduction these drugs in in vitro susceptibility testing, as susceptibility test-

ing fails to detect isolates with MIC values close to the breakpoint
Colistin (COL) became available for clinical use in the 1960s but [1,4-6]. Thus, a joint CLSI (Clinical and Laboratory Standards In-
other antibiotics replaced it 10 years later due to its toxicity [1]. stitute)/ EUCAST (European Committeee on Antimicrobial Suscep-
However, in the last few years, it has been re-introduced for man- tibility Testing) document recommends the use of broth dilution
aging infections by multidrug-resistant Gram-negative bacilli [2,3]. to perform susceptibility to COL [7]. Reference broth microdilution
The rise in the use of polymyxins, both in hospital and veterinary (BMD) is rarely performed by most clinical microbiology laborato-
settings, has led to an increase in the reports of acquired COL- ries because it is laborious. Several methods have been designed
resistant isolates [2]. This scenario has revealed the limitations of in the last few years, including rapid tests for Enterobacteriaceae,
which provide immediate results as the main advantage [8-10].
Due to the ability of Acinetobacter baumannii (A. baumannii)
∗
Corresponding author. Laboratorio de Bacteriología, Departamento de Bio-
to acquire multiple antibiotic resistance traits, the Infectious Dis-
química Clínica, Hospital de Clínicas José de San Martín, Facultad de Farmacia y
Bioquímica, Universidad de Buenos Aires, Buenos Aires, INFIBIOC, UBA, Argentina. eases Society of America has included it among the six pathogens
E-mail address: [email protected] (C.H. Rodriguez). resistant to antimicrobials (Enterococcus faecium, Staphylococcus

https://doi.org/10.1016/j.ijantimicag.2019.08.010
0924-8579/© 2019 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.
588 C.H. Rodriguez, G. Traglia and N. Bastias et al. / International Journal of Antimicrobial Agents 54 (2019) 587–591

aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Enter- 0.05% rezarzurine, with a pH adjusted to 7.4 ± 0.2. Tubes contain-
obacter spp) responsible for high morbidity and mortality in hospi- ing 175 μl of this solution without (control tube) and with COL
talised patients. Regarding the known mechanisms of resistance to (test tube) in a final concentration of 3.8 mg/L were inoculated
COL, opposed to what happens with Enterobacteriaceae, COL resis- with 25 μl of a bacterial suspension with a turbidity compatible
tance in A. baumannii is only chromosomic [1]. with 0.5 McFarland, and incubated for 3 hours at 35 °C. The test
Colistin resistance in A. baumannii is mediated by the addition result was positive when the test tube turned from purple to pink,
of phosphoethanolamine to the lipid A of lipopolysaccharide as a giving the same colour as the control tube, indicating growth in
result of mutations in the pmrAB two-component system or com- the presence of COL (COL resistance), whereas the test result was
plete loss of lipopolysaccharide expression due to mutations in the negative when the test tube remained purple (COL susceptible). A
lpxA, lpxC and lpxD genes [1,2]. Recently, efflux mechanisms have concentration of 3.8 mg/L was used as it is slightly lower than the
also been described [11]. Heteroresistance (HR) and COL depen- breakpoint. For comparison of methodologies performance, BDM
dence (CD) are also well-described phenomena in A. baumannii was taken as the reference method. Errors were ranked as follows:
clinical isolates. Heteroresistance is defined by growth of subpop- very major error, false susceptibility results by ADM, Pho, Pho24 or
ulations on plates containing 8 mg/L of colistin and CD as COL- rapid colistin Acinetobacter spp. (RCA); or major errors, false resis-
resistant subpopulations that grow in presence of a COL concen- tant results by ADM, Pho, Pho24 or RCA.
tration > 8 mg/L only near the COL disks [2,12-14]. Several authors
have defined the influence of mutations in pmrB and/or lpx genes 2.4. Population analysis
in growth rate, biofilm formation and fitness decrease [15,16]. Like-
wise, the regrowth observed in killing curves has been attributed Population analysis screening profiles were performed in dupli-
to resistant subpopulations in HR isolates [14,17,18]. However, there cate in the 165 isolates according to a previous report, with sev-
are no studies that have evaluated the impact of the two principal eral modifications [14]. Briefly, the Mueller-Hinton agar (Oxoid, UK,
COL-resistance mechanisms and the HR on the performance of the lot 2253971) plates with or without 6 mg/L of COL were inocu-
susceptibility methods. lated with 50 μl of cell suspension (0.5 Mc Farland). Colonies were
The present study aimed to determine the resistance mecha- counted after 48 hours of incubation at 35 °C. The frequency of re-
nisms in isolates with a COL MIC ≥ 4 mg/L and to correlate the sistant subpopulations (HR) was calculated by dividing the growth
mechanisms of resistance with the difficulties in detecting them (CFU) with 6 mg/L of COL by the growth (CFU) without COL.
with in vitro susceptibility testing. Furthermore, a rapid test to de-
tect COL resistance in A. baumannii was evaluated. 2.5. Time-kill curves

2. Materials and methods In five selected isolates with different MIC values and gene mu-
tations (2858 COL MIC 32 mg/L and lpx gene mutated; 8058 COL
2.1. Bacterial isolates and species identification MIC 32 mg/L and pmrB gene mutated; 52SJ COL MIC 8 mg/L and
lpxC gene mutated; 98726 COL MIC 4 mg/L and lpxD gene mutated;
A collection of 165 non-duplicated A. baumannii strains from and 269 COL MIC 4 mg/L and lpxD gene mutated) the growth
different clinical specimens of hospitalised patients was studied speed of resistant isolates with and without COL was evaluated by
during the period 2004–2017. A COL-dependent mutant (CAT-1D) time-kill studies. The studies were performed in duplicate. Tubes
obtained from A. baumannii strain (CAT-1), as previously described, containing cation-supplemented Mueller-Hinton broth (Oxoid, UK,
was also included [10]. The isolates were recovered from 15 hos- lot 1900545) with and without antibiotic were inoculated with
pitals in six South American countries. These isolates were iden- 5 × 105 CFU/mL of each isolate in log phase/mL. They were incu-
tified at the Hospital de Clínicas José de San Martín, Buenos Aires bated at 37 °C and killing was assessed at 0, 4, 6, 16 and 24 hours
city, using standard biochemical tests. Genospecies were confirmed by performing serial 10-fold dilutions and coating the aliquots onto
using matrix-assisted laser desorption and ionisation time-of-flight nutrient agar. The COL concentration was 3 mg/L because it was in-
mass spectrometry (MALDI-TOF MS), and/or by rpoB sequencing cluded in the concentration range of the Phoenix NMIC-406 panel.
[19]. The killing curves from the five isolates without COL were shown
as an average curve (T). Colistin sulfate (Sigma Aldrich, St Louis,
USA, lot 070M1499V) was used in all the methods performed in
2.2. Susceptibility testing this study.

The COL MICs were determined using the Mueller-Hinton broth 2.6. Detection of lpxACD and pmrB gene mutations
dilution method (BDM) (Oxoid, UK, lot 1900545), using glass tubes
during all procedures, and also by the agar dilution method (ADM) Detecting lpxACD and pmrB gene mutations in 17 A. bauman-
(Oxoid, UK, lot 2253971). Dilution methods were performed ac- nii isolates was performed by 16S ARN ribosomal amplification
cording to CLSI procedures. The COL susceptibility levels were also following capillary sequencing. Briefly: total DNA was extracted
determined by Phoenix 100 ID/AST system (Becton Dickinson Co., using ADN Puriprep B-kit (INBIO Highway, Argentina) and used
Sparks, Md.) using the Phoenix NMIC-406 panel, according to the as the template for PCR reactions, which were carried out us-
manufacture’s recommendations (Pho). In addition, a second read- ing the ‘Kit-T plus ADN polimerasa’ enzyme according to manu-
ing, by visual examination of the COL-containing wells of the pre- facturer’s instructions (INBIO Highway, Argentina), and the prod-
viously mentioned panel, was performed at 24 hours of incubation ucts were detected by agarose gel electrophoresis. The specific
(Pho24). A change from bluish to pink indicated bacterial growth. primers proposed by Moffatt et al. were used to analyse the pres-
Results were interpreted according to CLSI breakpoints [20]. ence of different mutations of lpxA, lpxC and lpxD genes [21]. The
COL-susceptible A. baumannii ATCC19606 strain (GenBank acces-
2.3. Rapid colistin Acinetobacter spp sion number ACQB010 0 0 0 0 0) was used as the reference sequence.
For detecting pmrB gene mutations, the specific primers reported
All of the isolates were subjected to a rapid colorimetric colistin by Beceiro et al. were used [22]. The A. baumanni ATCC 17978
sensitivity test. Briefly: 1 L of an alkaline solution was prepared strain (GenBank accession number CP0 0 0521.1) was included as
containing 10 g peptone, 5 g sodium chloride, 3 g beef extract, and the reference sequence. Sequencing was performed on both DNA
 C.H. Rodriguez, G. Traglia and N. Bastias et al. / International Journal of Antimicrobial Agents 54 (2019) 587–591 589

Table 1 tests; this was possibly due to the fact that resistant subpopula-
The MIC distribution, performance of the different colistin-susceptibility tests, and
tions are only able to grow after 24–48 h of incubation. However,
detection methods compared with BMD in the 165 isolates. Categorical agreement
analysis. in the 25 COL-resistant isolates, the performance varied according
to the COL MIC value. The number of errors in each method in the
BMD MIC Number of isolates Number of isolates (%)
resistant isolates was as follows: RCA four of 25; ADM eight of 25;
(μg/mL)
AD Pho Pho24 RCA Pho24 eight of 25, and Pho 13 of 25. The distribution of very major
≤ 0.5 16 16 (100) 16 (100) 16 (100) 16 (100) errors according to the MIC value is shown in Table 2.
1 59 59 (100) 59 (100) 59 (100) 59 (100) In order to determine the genetic basis of colistin resistance in
2 65 65 (100) 65 (100) 65 (100) 65 (100) A. baumannii, 17 randomly selected isolates were amplified and se-
4 6 0 0 0 2
quenced with different COL MICs. Nine different LpxA amino acid
8 2 0 0 0 2
16 4 4 3 4 4 changes over five mutant isolates, 10 different LpxC amino acid
32 9 9 5 9 9 changes over five mutant isolates, and 15 different LpxD amino
≥ 64 4 4 4 4 4 acid changes over nine mutant isolates were observed (Table 2).
AD, agar dilution; Pho, Phoenix NMIC-406; Pho24, Phoenix NMIC-406 manual read- Three isolates harboured LpxA mutation at 175 amino acid posi-
ing at 24 hrs of incubation; RCA, rapid colistin Acinetobacter spp. method; BMD, tion. Those three COL-resistant isolates contained the same amino
broth microdilution acid change (L175K). Isolates presented an amino acid change over
176 position. Two COL-resistant isolates contained the same muta-
tion at 176 position (I176S). Two COL-resistant isolates presented
strands using an ABIPrism 3100 BioAnalyzer and Taq FS Terminator
L175K and I176S amino acid changes with the same COL MIC. The
Chemistry (Taq FS, Perkin–Elmer). Sequences were examined and
presence of L175K and I176S mutations in the same isolate has
assembled with BioEdit software (version 7.0.5) and BLAST (version
no additive effect in the COL-resistant phenotype (Supplementary
2.6.0) software.
Table S1). All of the LpxC mutant isolates had N287D amino acid
3. Results and Discussion change. The mutation H264N was found in four COL-resistant iso-
lates. The Y260∗ mutation was found in two COL-resistant isolates.
The 165 clinical isolates were identified as A. baumannii by This mutation could have contributed to the increase in colistin
MALDI-TOF and rpoB gene sequencing. Twenty-five of these were MIC in A. baumannii. Other mutations in LpxC were found only in
COL resistant, as determined by the BMD method. The MIC50 COL-resistant isolates (Supplementary Table S1). Eight isolates had
and MIC90 values of the susceptible population were 1 mg/L and F311L amino acid changes to LpxD. Moreover, seven isolates con-
2 mg/L, respectively. A total of 46.7% of the susceptible isolates tained E312K amino acid changes to LpxD, being COL-resistant, and
presented an MIC of 2 mg/L. Fifteen COL-susceptible isolates were two with an MIC value close to the breakpoint (Supplementary Ta-
categorised as HR; 13 of 15 showed an MIC of 2 mg/L; 2 of 15 an ble S1). Five isolates contained a new mutation in the pmrB gene.
MIC of 1 mg/L; and 0 of 15 an MIC < 1 mg/L. The most prevalent mutation was T232I. Interestingly, all muta-
The correlations between the different methodologies and the tions were found in COL-resistant isolates (Supplementary Table
reference method are shown in Table 1. In COL-susceptible isolates, S1).
the agreement between the different methods was 100%. False In isolates with COL MIC ≥ 16 mg/L and with a high proportion
resistance was not detected in HR isolates in any of the performed of resistant subpopulations, two groups were found: one (n = 12)

Table 2
Epidemiological, genomic and susceptibility data for A. baumannii colistin-resistant isolates.

Isolate City Year MIC (μg/mL) Chromosomal mutations Accession number

BMD AD Pho Pho24 RAC HR LpxA LpxC LpxD PmrB

87 BA 2004 64 64 4 4 P 2 × 101 X MK533759

65027 BA 2005 4 1 ≤ 1 ≤ 1 N 1 × 10−5 X MK533760
214 BA 2013 4 1 ≤ 1 ≤ 1 N 3 × 10−5 X MK533761
220 BA 2013 4 2 ≤ 1 ≤ 1 N 2 × 10−5 X MK561297
259 BA 2014 16 16 4 4 P 2 × 101 ND ND ND ND
406 BA 2015 64 64 4 4 P 2 × 101 ND ND ND ND
86800 BA 2015 16 16 4 4 P 2 × 101 ND ND ND ND
4840 Mendoza 2016 32 32 2 4 P 4 × 101 X X MK561298/MK561299
3662 Mendoza 2016 16 16 2 4 P 2 × 101 X MK561300
2858 Mendoza 2016 32 32 4 4 P 1 × 101 X X MK621809/MK62810
4888 Mendoza 2016 16 16 4 4 P 2 × 101 X X MK621811/MK621812
8058 Mendoza 2016 32 32 2 4 P 3 × 101 X MK621813
1993 Mendoza 2016 32 32 4 4 P 1 × 101 X MK621814
2320 Mendoza 2016 32 32 4 4 P 2 × 101 X MK621815
2315 Mendoza 2016 32 32 4 4 P 1 × 101 ND ND ND ND
4395 Mendoza 2016 32 32 2 4 P 1 × 101 ND ND ND ND
6218 Mendoza 2016 32 32 4 4 P 2 × 101 X X MK621816/MK621817
2521 Mendoza 2016 32 32 2 4 P 3 × 101 X X X X MK621818/MK621819
52SJ San Juan 2016 8 2 ≤ 1 ≤ 1 P 2 × 10−3 X MK533762
53SJ San Juan 2016 4 2 ≤ 1 ≤ 1 P 5 × 10−4 ND ND ND ND
85 Chaco 2016 8 0.5 ≤ 1 ≤ 1 P 5 × 10−5 X MK860815
98726 BA 2017 4 2 ≤ 1 ≤ 1 P 4 × 10−4 X MK860813
269 BA 2017 4 1 ≤ 1 ≤ 1 N 8 × 10−4 X MK860814
47 BA 2018 64 32 4 4 P 1 × 101 ND ND ND ND
270 BA 2018 64 32 4 4 P 2 × 101 ND ND ND ND

AD, agar dilution; Pho, Phoenix NMIC-406; Pho24, Phoenix NMIC-406 manual reading at 24 h of incubation; RCA, rapid colistin Acinetobacter spp. method; HR, frequency of
resistant subpopulations; BA, Buenos Aires city; P, positive; N, negative; ND, not determined
Bold type, very major errors
590 C.H. Rodriguez, G. Traglia and N. Bastias et al. / International Journal of Antimicrobial Agents 54 (2019) 587–591

Fig. 1. Time-kill studies performed on five colistin-resistant Acinetobacter baumannii isolates.

was correctly categorised by Pho and Pho24, and the other (n = 5) ter spp., which is easier to perform than other tests that have been
was wrongly categorised by Pho, but was corrected when read at recently described [10].
24 h (Pho24). The isolates in the first group harboured heteroge- Acinetobacter baumannii COL-dependent isolates have been de-
neous mutations in lpxA and lpxC but not in pmrB. As observed in scribed in the literature but their presence in clinical isolates is
Fig. 1, the isolate 2858 (representative of the first group), which sporadic. The mutant isolate with COL MIC > 64 mg/L included in
was correctly categorised by Pho, showed a killing curve similar to the current study did not show growth in the automatised system,
the T curve. Conversely, the isolates in the second group harboured and in the RCA test it was only able to grow in the tube contain-
mutations in the pmrB; the resistant CFUs showed a slow growth ing COL. As previously mentioned, major errors were not detected
between 4 and 16 h. This delay in the growth of resistant subpop- and the growth of resistant subpopulations in the HR isolates was
ulations could be the reason why the Phoenix System is unable to prevented by the early reading. As previously described by other
detect them, as its final reading is performed at 10–12 h of incu- authors [4,6], most of the errors were observed in isolates with
bation. The fact that all the isolates belonging to this group were MIC values close to the breakpoint.
correctly categorised by Pho24 reinforces the idea that the delay
in growth speed is the main source of error. No differences were 4. Conclusion
found when comparing the reference method and ADM in isolates
with an MIC ≥ 16 mg/L. There are multiple reasons to explain the failures in detect-
There was no agreement between Pho and BMD in isolates ing COL resistance. The isolates that were correctly categorised
with an MIC range of 4–8 mg/L and reading the panel at 24 h showed high MIC values (> 16 mg/L), a high proportion of resis-
did not improve detection in these cases. In these isolates (52SJ, tant subpopulations, and a growth speed that did not vary in the
98726 and 269), growth was slower and there was an increase presence or absence of COL. While there are less of these char-
in the resistant CFUs after 16 h of incubation. Most of these iso- acteristics present in COL-resistant isolates, it is more difficult to
lates (seven of eight) carried mutations in lpxD. Automated sys- accurately detect them. The slow growth in isolates carrying mu-
tems may also fail to detect these COL-resistant isolates due the tations in lpxC is notorious. This phenomenon is also observed,
low proportion of resistant subpopulations (1 × 10−4 –1 × 10−5 ). to a lesser extent, in isolates with mutations in the pmrB gene.
This low frequency would result in 25 COL-R CFU encoding to Heteroresistant subpopulations would also play a vital role in the
the inoculum introduced in the panel under the conditions de- misdetection and difficulties in observing the final reading point.
scribed by the manufacturer; this CFU is 10 0 0 0 0 times lower Other factors that should also be mentioned are the proximity be-
compared with an homogeneously resistant isolate. Previous re- tween the breakpoint and the wild type median MIC value.
ports have shown similar results between BDM and ADM [23].
The current study observed discrepancies in eight borderline iso- Declarations
lates. Similar results were described by Dafopoulou [4]. The low
proportion of resistant subpopulations (between 20–0.2 CFU/mL at Declaration of Competing Interest
4 mg /L) in agar dilution spot (104 ) could lead to the misdetec-
tion of these isolates. Six of seven isolates showed mutations in lpx There are no conflicts of interest.
genes.
In the present study, RCA showed the best performance. Dis- Funding
crepancies were observed in four of six isolates with a COL MIC
4 mg/L; this was an expected result as the concentration used in This work was supported by Secretaría de Ciencia y Técnica de
the test was very close to the MIC value. RCA is an in-house rapid la Universidad de Buenos Aires (UBACyT 20020130100167BA) to
colorimetric method for detecting colistin resistance in Acinetobac- Ángela Famiglietti.
 C.H. Rodriguez, G. Traglia and N. Bastias et al. / International Journal of Antimicrobial Agents 54 (2019) 587–591 591

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