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Journal of Microbiological Methods 167 (2019) 105765

Contents lists available at ScienceDirect

Journal of Microbiological Methods


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

Note

An alternative disk diffusion test in broth and macrodilution method for T


colistin susceptibility in Enterobacteriales
López-Jácome Luis Esaúa, Rengel-Garcia Christian Rodolfoa, Hernández-Durán Melissaa,

Colín-Castro Claudia Adrianaa, García-Contreras Rodolfob, Franco-Cendejas Rafaela,
a
Infectious Diseases Division, Instituto Nacional de Rehabilitación Luis Guillermo Ibarra Ibarra, Mexico City, Mexico
b
Bacteriology Laboratory, Microbiology and Parasitology Department, Medicine Faculty, Universidad Nacional Autónoma de México, Mexico City, Mexico

A R T I C LE I N FO A B S T R A C T

Keywords: Colistin and polymyxin B are old drugs that have been reintroduced to treat Gram-negative infections lacking
Resistance other treatment options; however, colistin resistance have been reported. To know the correct susceptibility
Antibiotics pattern is mandatory for multidrug resistant bacteria. Broth microdilution method is the gold standard to
Enterobacteriales evaluate colistin and polymyxin B susceptibility; nevertheless, it is time consuming and needs expertise to be
Antimicrobials and infection
performed. Disk diffusion method on Müeller-Hinton agar is no longer recommended to evaluate polymyxins
Alternative methods
susceptibility. In this study we evaluated two methods (disk diffusion in broth and broth macrodilution) as
alternative options to identify polymyxin resistance in an easy way. A total of 536 Enterobacteriales isolates were
assessed for colistin susceptibility. All non-wild type Enterobacteriales (41) were chosen and 31 wild type bacteria
were randomly selected, were used to perform disk diffusion tests in broth and for broth macrodilution tests. We
found 100% of concordance between both tested methods and broth microdilution.
In conclusion, these two methods are reliable and easier options that complement as initial screening sus-
ceptibility for colistin in Enterobacteriales in microbiology laboratories lacking personnel and infrastructure to
perform broth microdilution method.

1. Introduction change, primarily by colistin poor diffusion on agar media (Poirel et al.,
2017; Velkov et al., 2010). The gold standard method is the broth mi-
Polymyxins, including polymyxin B and colistin, are lipopeptide crodilution test and international standards organizations such as the
antibiotics produced by the bacterium Paenibacillus polymyxa (Yahav Clinical and Laboratory Standards Institute (M100, 2018) and the
et al., 2012). They selectively bind to the lipopolysaccharide (LPS) of European Committee on Antimicrobial Susceptibility Testing (EUCAST,
Gram-negative bacteria, and also, they displace divalent cations that 2017.), recommend do not to perform it using gradient tests, agar disk
bridge adjacent LPS molecules inducing expansion of the outer bacterial diffusion or semi-automated devices such as Vitek 2®, Phoenix® or
membrane and loss of integrity of the inner membrane. Bacterial death MicroScan® due of their unreliable results. Most of microbiology la-
comes as a consequence of leakage of the intracellular content (Baron boratories around the world lack of trained staff and enough time to
et al., 2016; Hancock and Chapple, 1999). The increase of multidrug perform this particular methodology (Gwozdzinski et al., 2018;
resistant Gram-negative bacteria has promoted the reuse of these an- Matuschek et al., 2017). Optional techniques, such as macrodilution
tibiotic family members as a last resource option to treat patients. methods, have been accepted as adequate tests to evaluate suscept-
Nonetheless, resistance to this drugs in Gram-negative bacteria has ibility in microorganisms such as in Pseudomonas aeruginosa (Turlej-
been reported and it is mediated by chromosomal mutations and by Rogacka et al., 2018).
genes located in mobile elements, such as mcr-1 (Landman et al., 2008; The aim of this study was to test the macrodilution method and a
MacNair et al., 2018; Peterson et al., 1985). Laboratory tests to assure modified disk diffusion method as alternatives to screen colistin sus-
susceptibility or resistance to polymyxins have been in a continuous ceptibility in Enterobacteriales, both methods were compared with

Abbreviations: LPS, Lipopolysaccharide; CLSI, Clinical and Laboratory Standards Institute; EUCAST, European Committee on Antimicrobial Susceptibility Testing;
MIC, Minimal inhibitory concentration; Bmd, Broth microdilution; BMD, Broth macrodilution

Corresponding author at: Instituto Nacional de Rehabilitación Luis Guillermo Ibarra Ibarra. Av. México-Xochimilco #289 Col. Arenal de Guadalupe, Mexico City,
Mexico.
E-mail address: [email protected] (F.-C. Rafael).

https://doi.org/10.1016/j.mimet.2019.105765
Received 22 May 2019; Received in revised form 23 October 2019; Accepted 26 October 2019
Available online 30 October 2019
0167-7012/ © 2019 Elsevier B.V. All rights reserved.
L.-J. Luis Esaú, et al. Journal of Microbiological Methods 167 (2019) 105765

microdilution broth such as gold standard. resistant control. Each test for clinical and control samples were done
by triplicate. Non-wild type result was defined as optical turbidity, wild
2. Material and methods type result was defined as turbidity absence, both were read at 18 h.
To know the MIC by this method we used 10 μg colistin disks (BD
2.1. Bacterial strains BBL, USA) to prepare 10 dilutions, starting at a concentration of
64 μgmL−1, prepared in MH broth adjusted with cations, adding co-
536 clinical strains from Enterobacteriales were selected from a seven listin discs and incubated at 37 °C for 1 h to allow the diffusion of co-
years period (2011–2017). We performed the broth microdilution sus- listin. After this time, double dilutions of concentrations from 64 to
ceptibility method for colistin (Colistin sulfate salt, Sigma Aldrich C- 0.125 μg.mL−1 and a growth control were performed, according to the
4461. USA) to each isolate as gold standard and comparison method in CLSI M07 (M07, 2015) and CLSI M100 (M100, 2018). Bacterial sus-
order to validate our experimental assays according to CLSI M07 2018 pensions were made by adjusting them to 0.5 McFarland; 6.6 μL of this
methods for dilution antimicrobial susceptibility test for bacteria that suspension was taken and deposited in the saline tubes (1:150 dilution)
grow aerobically (M07, 2015). Susceptibility interpretation was made 5 × 105 CFU.mL−1. Subsequently, 1 mL of the different dilutions of
according to M100 Performance standards for antimicrobial suscept- colistin with MH was added, in descending form (64–0.125 μg.mL−1)
ibility testing (M100, 2018). All non-wild type Enterobacteriales isolates and ending with the growth control. P. aeruginosa ATCC 27853 and E.
were included, and we randomly selected (https://www.random.org/ coli ATCC 25922 were used as sensitive controls and P. vulgaris ATCC
lists/) the same number of wild-type microorganisms to compare them. 6380 and S. marcescens AC 17–1291 as resistant controls. They were
Bacteria distribution were as follow: 33 Enterobacter cloacae, 21 Es- incubated for 18–24 h. The MIC result was visually defined in the
cherichia coli, 16 Klebsiella pneumoniae and 2 Klebsiella aerogenes strains. concentration where turbidity was no longer observed and classified
All determinations were made by triplicate. according to the CLSI M100 (M100, 2018)guidelines in wild or non-
wild type.
2.2. Broth macrodilution method screening To confirm colistin susceptibility in the wild-type strains, we cen-
trifuge the broth from the macro-dilution test and the disk diffusion test
We decided to use only one concentration such as screening test. in broth and then we growth the pellet on blood agar plates to corro-
Briefly, we prepared colistin aliquots with a final concentration of borate bacteria viability.
4 μg.mL−1 (Colistin sulfate salt, Sigma Aldrich C-4461. USA) with
Müeller-Hinton (MH) broth supplemented with cations as CLSI refers
(M100, 2018). Aliquots were kept at −20 °C until its use. In a 5 mL 2.4. Bacterial DNA extraction
tube, 1 mL of isotonic saline solution was verted and then 6.6 μL were
taken. Bacterial suspension was adjusted at 0.5 of McFarland scale. DNA extraction of all strains, wild type and Non-wild type, were
From that suspension 6.6 μL were taken and deposited into saline so- performed by thermal shock using Chelex resin (BioRad, USA). It was
lution. Afterwards, 1 mL of MH broth cation adjusted with 4 μg.mL−1 of carried out taking two colonies of the pure culture and dissolved in
colistin was added to have a final concentration of 5 × 105 UFC.mL−1 100 μL of Chelex 5%, the vial was placed at 95 °C for 30 min, then
and a final colistin concentration of 2 μg.mL−1. P. aeruginosa ATCC centrifuged at 9600g for 5 min. The supernatant was quantified in
27853 was used as susceptible control and Proteus vulgaris ATCC 6380 NanoDrop (Thermo Fischer, USA) and a dilution was made until a final
as intrinsically resistant control. Every sample and control were done by concentration of 20 ng.μL−1 was obtained.
triplicate assays. Non-wild type result was defined as optical turbidity
visually, wild type result was defined as turbidity absence, and both 2.5. mcr-1 amplification
were read after 18 h of incubation at 37 °C.
In order to know the colistin MIC we prepared 10 double dilutions It was performed by using PCR endpoint; briefly: 25 ng of DNA,
from 0.125 to 64 μg.mL−1 on MH broth adjusted with cations, as re- 10 mmol L−1 of dntp's, 1 mmol L−1 of MgCl2, 10 pmol of each oligo-
ferred to in CLSI M07 and CLSI M100 (M100, 2018). 11 tubes of 5 mL nucleotide (mcr-1-F 5′CGGTCAGTCCGTTTGTTC-3′ and mcr1-R 5′-CTT
were taken, 1 mL of isotonic saline was placed for each dilution and a GGTCGGTCTGTAGGG-3′)(Chang et al., 2017), a unit of AmpliTaq Gold
tube for growth control, 6.6 μL was removed. Bacterial suspensions (Thermo Fisher Scientific, USA). Program conditions were: 94 °C for
were made by adjusting them to 0.5 McFarland; 6.6 μL of this suspen- 5 min, followed by 35 cycles of 94 °C 30 s, 55 °C 30 s and 72 °C for 40 s,
sion was taken and deposited in the saline tubes (dilution 1: 150). finally 72 °C for 10 min, the thermocycler used was Veriti (Applied
Subsequently, 1 mL of the different dilutions of colistin with MH was Biosystems, USA). Positive control was kindly given by Instituto Na-
added, in descending form (64–0.125 μg / mL) and ending with the cional de Salud Pública (Garza-Ramos et al., 2018). The 335 bp PCR
growth control. P. aeruginosa ATCC 27853 and E. coli ATCC 25922 were products were visualized on a 1% agarose gel, using SYBR Green 1 as an
used as sensitive controls and Proteus vulgaris ATCC 6380 and S. mar- intercalating agent.
cescens AC 17–1291 as resistant controls; they were incubated from 18
to 24 h. MIC interpretation was defined visually, in the concentration
where turbidity was no longer observed and classified according to the 3. Results
CLSI M100 (M100, 2018) guidelines in wild or non-wild type.
We found 41 Enterobacteriales isolates with colistin
2.3. Disk diffusion in broth method MIC > 2 μg.mL−1 during the study period. When we performed the disk
diffusion in broth method, it showed 100% concordance with gold
10 μg colistin disks (BD BBL, USA) were used to perform the test. A standard, both wild type and non-wild type results. Macrodilution
5 mL tube was filled with 2.5 mL of isotonic saline solution, then method showed the same results as it was observed with the disk dif-
16.7 μL were removed. A colistin disk was set and then the tubes were fusion in broth method. The MIC verification of both methods was
incubated at 37 °C for 1 h to allow colistin diffusion. Bacterial suspen- equal as broth microdilution method when it was used as screening test,
sion was adjusted at 0.5 of McFarland scale and 16.7 μL were deposited none result was different; however, the final MIC in both methods
to the tube previously incubated. Then, 2.5 mL of MH broth were added showed positive differences compared to broth microdilution (Table 1).
with a final concentration of 2 μg.mL−1 of colistin in each tube having a We performed mrc-1 PCR in order to identify genetic resistance to co-
final concentration of 5 × 105 UFC.mL−1. P. aeruginosa ATCC 27853 listin; however, there were not positive results in susceptible or re-
was used as susceptible control strain and P. vulgaris ATCC 6380 as sistant samples.

2
L.-J. Luis Esaú, et al. Journal of Microbiological Methods 167 (2019) 105765

Table 1
Colistin susceptibility results among the three different tests in Enterobacteriales strains.
Strain Bacteria MIC-Bmd MIC-BMD (μg.mL−1) MIC – BMD Verification MIC-Disk (μg.mL−1) MIC – Disk Verification Interpretation
(μg.mL−1) screening (μg.mL−1) screening (μg.mL−1)

EB116 E. cloacae 1 ≤2 0.5 ≤2 0.5 Wild type


EB773 E. cloacae 1 ≤2 0.5 ≤2 0.5 Wild type
EB1581 E. cloacae 1 ≤2 0.5 ≤2 0.5 Wild type
EB1822 E. cloacae 1 ≤2 0.5 ≤2 0.5 Wild type
EB1823 E. cloacae 1 ≤2 0.5 ≤2 0.5 Wild type
EB1825 E. cloacae 1 ≤2 0.5 ≤2 0.5 Wild type
EB1855 E. cloacae 1 ≤2 0.5 ≤2 0.5 Wild type
EB1986 E. cloacae ≤0.25 ≤2 0.25 ≤2 0.25 Wild type
EB2234 E. cloacae 1 ≤2 0.5 ≤2 0.5 Wild type
EB2779 E. cloacae 1 ≤2 0.5 ≤2 0.5 Wild type
EB3115 E. cloacae 1 ≤2 0.5 ≤2 0.5 Wild type
EB3119 E. cloacae 1 ≤2 0.5 ≤2 0.5 Wild type
EB3123 E. cloacae 1 ≤2 0.5 ≤2 0.5 Wild type
EB3128 E. cloacae 1 ≤2 1 ≤2 2 Wild type
EB3704 E. cloacae ≤0.25 ≤2 0.5 ≤2 0.5 Wild type
EB3747 E. cloacae ≤0.25 ≤2 0.5 ≤2 0.5 Wild type
EB443 E. coli 0.5 ≤2 0.5 ≤2 0.5 Wild type
EB791 E. coli 0.5 ≤2 0.5 ≤2 0.25 Wild type
EB840 E. coli ≤0.25 ≤2 0.25 ≤2 0.25 Wild type
EB3669 E. coli ≤0.25 ≤2 0.125 ≤2 0.25 Wild type
EB3671 E. coli ≤0.25 ≤2 0.125 ≤2 0.125 Wild type
EB3681 E. coli ≤0.25 ≤2 0.125 ≤2 0.125 Wild type
EB3722 E. coli ≤0.25 ≤2 0.125 ≤2 0.125 Wild type
EB270 K. aerogenes ≤0.25 ≤2 0.125 ≤2 0.125 Wild type
EB1719 K. aerogenes 1 ≤2 0.5 ≤2 0.5 Wild type
EB365 K. pneumoniae ≤0.25 ≤2 0.125 ≤2 0.125 Wild type
EB478 K. pneumoniae ≤0.25 ≤2 0.125 ≤2 0.125 Wild type
EB628 K. pneumoniae ≤0.25 ≤2 0.25 ≤2 0.125 Wild type
EB3698 K. pneumoniae ≤0.25 ≤2 0.125 ≤2 0.125 Wild type
EB3733 K. pneumoniae ≤0.25 ≤2 0.25 ≤2 0.25 Wild type
EB3751 K. pneumoniae ≤0.25 ≤2 0.25 ≤2 0.25 Wild type
EB001 E. cloacae ≥ 64 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB024 E. cloacae 0.125 <2 ≤0.125 <2 ≤0.125 Non-wild type
EB043 E. cloacae > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB081 E. cloacae > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB487 E. cloacae > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB1502 E. cloacae 8 >2 8 >2 8 Non-wild type
EB1575 E. cloacae > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB1604 E. cloacae > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB1635 E. cloacae 16 >2 16 >2 16 Non-wild type
EB1672 E. cloacae > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB1899 E. cloacae > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB1977 E. cloacae 16 >2 16 >2 16 Non-wild type
EB1981 E. cloacae 16 >2 16 >2 16 Non-wild type
EB2074 E. cloacae > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB2216 E. cloacae > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB2898 E. cloacae > 2 >2 4 >2 4 Non-wild type
EB3020 E. cloacae > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB300 E. coli > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB566 E. coli > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB581 E. coli > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB791 E. coli 8 >2 8 >2 8 Non-wild type
EB840 E. coli 8 >2 8 >2 8 Non-wild type
EB1234 E. coli > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB1407 E. coli > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB1568 E. coli > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB1603 E. coli > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB1934 E. coli > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB2075 E. coli > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB2541 E. coli 4 >2 4 >2 4 Non-wild type
EB2544 E. coli > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB2891 E. coli > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB478 K. pneumoniae 8 >2 8 >2 8 Non-wild type
EB882 K. pneumoniae > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB927 K. pneumoniae 4 >2 4 >2 4 Non-wild type
EB1105 K. pneumoniae 8 >2 8 >2 8 Non-wild type
EB1337 K. pneumoniae > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB1432 K. pneumoniae > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB1602 K. pneumoniae > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB1791 K. pneumoniae > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB2394 K. pneumoniae > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
EB2761 K. pneumoniae > 2 >2 ≥ 64 >2 ≥ 64 Non-wild type
ATCC 27853 P. aeruginosa 0.5 ≤2 0.5 ≤2 0.5 Susceptible
(continued on next page)

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L.-J. Luis Esaú, et al. Journal of Microbiological Methods 167 (2019) 105765

Table 1 (continued)

Strain Bacteria MIC-Bmd MIC-BMD (μg.mL−1) MIC – BMD Verification MIC-Disk (μg.mL−1) MIC – Disk Verification Interpretation
(μg.mL−1) screening (μg.mL−1) screening (μg.mL−1)

ATCC 25922 E. coli 0.25 <2 0.25 <2 0.25 Wild type
CI 17–1291 S. marcescens ≥ 64 >2 ≥64 >2 ≥ 64 Intrinsically resistant
ATCC 6380 P. vulgaris 8 >2 8 >2 8 Intrinsically resistant

MIC-Bmd: Minimal inhibitory concentration broth microdilution; MIC-BMD: Minimal inhibitory concentration broth macrodilution method; MIC-Disk: Minimal
inhibitory concentration broth diffusion disk. CI, confirmed linical isolate.

4. Discussion Declaration of Competing Interest

Gram-negative multidrug resistance is a dynamic and emerging The authors declare that there is no conflict of interest.
public health problem around the world. Colistin in several cases is the
last resource antibiotic; however, physicochemical characteristics re- Acknowledgements
duce the possibility to do simple susceptibility tests. The most com-
monly used method in routine laboratories, disk diffusion, has been We thank to Dr. Celia Alpuche Aranda and Dr. Jesús Silva Sánchez
shown to be unreliable due to poor colistin diffusion in agar. Recently, from Instituto de Salud Pública de México, who kindly gave to us mcr-1
CLSI and EUCAST suggest performing microdilution broth susceptibility positive control.
method in order to get reliable results. Enterobacteriales infections are a
global concern, and some reports show the presence of resistance to References
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having an unfailing test might be giant.
Having reliable and easy tests for colistin susceptibility different to
broth microdilution is important for all clinical microbiology labora-
tories in order to assess its clinical utility as another consistent option.

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