Journal of Microbiological Methods 90 (2012) 160166

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Journal of Microbiological Methods

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

Comparison of the WST-8 colorimetric method and the CLSI broth microdilution
method for susceptibility testing against drug-resistant bacteria
Tadayuki Tsukatani a,, Hikaru Suenaga a, Masanobu Shiga b, Katsuya Noguchi b, Munetaka Ishiyama b,
Takatoshi Ezoe b, Kiyoshi Matsumoto c
a
b
c

Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center, Kurume 8390861, Japan
Dojindo Laboratories, Kumamoto 8612202, Japan
Laboratory of Food Bioscience, Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Kumamoto 8600082, Japan

a r t i c l e

i n f o

Article history:
Received 8 February 2012
Received in revised form 1 May 2012
Accepted 4 May 2012
Available online 27 May 2012
Keywords:
Drug-resistant
Electron mediator
Microorganism
Naphthoquinone
Susceptibility testing
Tetrazolium salt

a b s t r a c t
The minimum inhibitory concentrations (MICs) obtained from the susceptibility testing of various bacteria to
antibiotics were determined by a colorimetric microbial viability assay based on reduction of a tetrazolium
salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium
salt (WST-8)} via 2-methyl-1,4-napthoquinone as an electron mediator and compared with those obtained
by the broth microdilution methods approved by the Clinical and Laboratory Standard Institute (CLSI). Especially for drug-resistant bacteria, the CLSI method at an incubation time of 24 h tended to give lower MICs.
The extension of incubation time was necessary to obtain consistent MICs for drug-resistant bacteria such
as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococi (VRE) and multidrug resistant Pseudomonas aeruginosa (MDRP) in the broth microdilution method. There was excellent
agreement between the MICs determined after 24 h using the WST-8 colorimetric method and those
obtained after 4896 h using the broth microdilution method. The results suggest that the WST-8 colorimetric
assay is a useful method for rapid determination of consistent MICs for drug-resistant bacteria.
2012 Elsevier B.V. All rights reserved.

1. Introduction
The prevalence of clinical infections caused by drug-resistant microorganisms continues to increase throughout the world although medical and sanitary efforts have advanced. Recently, in addition to wellknown drug-resistant microorganisms such as methicillin-resistant
Staphylococcus aureus (MRSA) (Hsueh et al., 2004; Moran et al., 2006),
vancomycin-resistant enterococi (VRE) (Deshpande et al., 2007), and
extended spectrum -lactamase (ESBL)-producing Enterobacteriaceae
(Pitout and Laupland, 2008; Valverde et al., 2004), new infections due
to multidrug-resistant bacteria have been increasing. Infections due to
multidrug-resistant Pseudomonas aeruginosa (MDRP) (Gaynes and
Edwards, 2005; Pagani et al., 2005; Sekiguchi et al., 2007), multidrugresistant Acinetobacter baumannii (Dijkshoorn et al., 2007; Gaynes and
Edwards, 2005; Higgins et al., 2010), and carbapenemase-producing
Klebsiella pneumoniae (Bratu et al., 2005; Leavitt et al., 2007;
Nordmann et al., 2009) have rapidly emerged. Furthermore, gramnegative Enterobacteriaceae with multidrug-resistance caused by New
Delhi metallo--lactamase 1 (NDM-1) are potentially a major global

Corresponding author at: Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center, 14655 Aikawamachi, Kurume 8390861, Japan. Tel.:
+ 81 942 30 6644; fax: + 81 942 30 7244.
E-mail address: tukatani@tc.pref.fukuoka.jp (T. Tsukatani).
0167-7012/$ see front matter 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2012.05.001

health problem (Kumarasamy et al., 2010; Nordmann et al., 2011;

Yong et al., 2009).
Therefore, rapid and accurate antimicrobial susceptibility testing
is increasingly important for appropriate patient management and
clinical surveillance. In general, when any type of antimicrobial susceptibility testing is performed in the clinical laboratory, a standard
method approved by the Clinical and Laboratory Standard Institute
(CLSI) (CLSI, 2006) is frequently used. In the broth microdilution
method recommended by CLSI, the incubation time is set between
16 and 20 h for determining the antimicrobial susceptibility for pathogenic bacteria. However, the incubation time may differ for some
problem microorganisms possessing resistance which is difcult to
detect (CLSI, 2006). In this CLSI document, it is indicated that accurate
detection of methicillin-resistant staphylococci and VRE requires incubation for a full 24 h rather than 16 to 20 h (CLSI, 2006). It has
been reported that the extension of the incubation period to 48 h signicantly increased the detection rate of methicillin-resistant staphylococci (Aldridge et al., 1983; Boyce et al., 1984; Woods et al., 1986).
The possibility of enhancing the detection of VRE by extending the incubation time to 48 h has been also reported (Sahmi and Olsen,
1990). These reports suggested that the MICs determined by the microdilution method increased with increasing incubation time for
these drug-resistant bacteria. Thus, extension of the incubation time
might be necessary to obtain the consistent minimum inhibitory concentration (MIC) for drug-resistant bacteria.

T. Tsukatani et al. / Journal of Microbiological Methods 90 (2012) 160166

Methods for rapid susceptibility testing using tetrazolium salts as indicator reagents have been developed (Brady et al., 2007; Meletiadis et
al., 2001; Moriartya et al., 2005; Tunney et al., 2004). The most commonly used tetrazolium salt in colorimetric assays for microorganism
testing has been 2,3-bis (2-methyloxy-4-nitro-5-sulfophenyl)-5[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT), which,
after reduction, yields a water-soluble formazan derivative that can be
easily quantied colorimetrically (Meletiadis et al., 2001; Paull et al.,
1988; Tunney et al., 2004). However, we have reported that XTT is easily reduced by culture media components such as peptones and glycated proteins or some antibiotics (Tsukatani et al., 2009). The noncellular reduction of tetrazolium salts leads to an underestimation of
the activity of antimicrobial substances. When XTT was employed, the
non-cellular reductions in MuellerHinton broth in the absence of microorganisms were marked. Furthermore, the non-cellular reduction
of XTT was promoted by the addition of antibiotics. We have developed
a colorimetric method based on the reduction of the tetrazolium salt {2(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)2H-tetrazolium, monosodium salt (WST-8)} for a microbial viability
assay (Tsukatani et al., 2008, 2009). MuellerHinton broth gave minimal rise to the non-cellular reduction of WST-8. Our results indicated
that WST-8 was superior to XTT in terms of its reactive efciency with
the electron mediators (reduced form) produced by microorganisms
and of its tolerance to medium components. In this method, 2methyl-1,4-naphthoquinone (NQ) used as an electron mediator was reduced by microorganisms, and WST-8 was then reduced by the produced naphthohydroquinone to its formazan which exhibits a
maximum absorbance at 460 nm.
The purpose of this study is to apply the WST-8 colorimetric method to antimicrobial susceptibility testing for various drug-resistant
bacteria and to demonstrate the advantages of the present method
as compared to the broth microdilution methods approved by CLSI.
The WST-8 colorimetric method would provide a useful means for
the rapid determination of consistent MICs for several drugresistant bacteria.
2. Materials and methods
2.1. Chemicals and media
2-Methyl-1,4-NQ was obtained from Sigma Chemicals (St. Louis,
MO, USA). 2-(2-Methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) was a gift
from Dojindo (Kumamoto, Japan). Cation-adjusted MuellerHinton
broth and Haemophilus test medium (HTM) were purchased from
Kyokuto Pharmaceutical Industrial Co. (Tokyo, Japan). Tryptic soy
broth, yeast extract, and de ManRogosaSharpe (MRS) media were
obtained from Difco Laboratories (Detroit, MI, USA). All other
chemicals were of analytical reagent grade and were used without further purication.
2.2. Detection reagents
2-Methyl-1,4-NQ as an electron mediator was dissolved in dimethyl sulfoxide (DMSO) at concentrations of 1.0 mM. WST-8 was
dissolved in distilled water at a concentration of 11.1 mM and the solution was sterilized by passing it through a cellulose acetate membrane lter (pore size, 0.2 m; diameter, 13 mm). The tetrazolium
salt solution was mixed with electron mediators at a ratio of 9:1.
The prepared detection reagent contained 10 mM WST-8, 0.1 mM 2methyl-1,4-NQ, and 10% DMSO.

161

(PCG), piperacillin (PIPC)), cephems (cexime (CFIX), cefotaxime

(CTX), ceftazidime (CAZ)), monobactams (aztreonam (ATZ)), penems
(imipenem (IPM)), aminoglycosides (amikacin (AMK), gentamicin
(GM)), quinolones (ciprooxacin (CPFX), levooxacin (LVFX)),
lipopeptides (colistin (CL)), macrolides (clarithromycin (CAM)), glycopeptides (vancomycin (VCM)), phenicols (chloramphenicol (CP)),
tetracyclines (tetracycline (TC)).
2.4. Microbial strains and growth conditions
Bacteria used in this study were obtained from the Biological Resource Center at the National Institute of Technology and Evaluation
(NBRC, Chiba, Japan), the Japan Collection of Microorganisms, RIKEN
BioResource Center (JCM, Tsukuba, Japan), the American Type Culture
Collection (ATCC, Rockville MD, USA), and Gifu University (Gifu,
Japan). Enterococcus faecalis ATCC51299vr (VRE) was produced from
E. faecalis ATCC51299 by repeated daily passage in MRS containing increasing levels of vancomycin up to 128 g/ml. Batches of medium
(5 ml in a glass test tube) were inoculated from fresh culture plates
and incubated for 18 h at the optimum temperature (30 or 37 C).
The culture media contained tryptic soy broth plus yeast for all the
bacteria except HTM (MuellerHinton broth with 0.5% yeast extract,
1.5 mg% hemin and 1.5 mg% NAD) was used for Haemophilus sp. and
MRS was used for Enterococcus sp.
2.5. Susceptibility testing
Reference MICs were determined by the broth microdilution
method currently recommended by CLSI (CLSI, 2006). Serial twofold dilutions of each antibiotic were prepared in cation-adjusted
MuellerHinton broth or HTM. Cation-adjusted MuellerHinton
broth was used for all bacteria with the exception of Haemophilus
sp., Neisseria sp. and Streptococcus sp. HTM was used for Haemophilus
sp. For Neisseria sp. and Streptococcus sp., cation-adjusted Mueller
Hinton broth containing 0.3% horse blood was employed. MPIPC and
VCM were used as the reference antibiotics for MRSA and VRE, respectively. PIPC, CAZ, CPFX, ATZ, IPM, AMK and GM were employed
for MDRP. CAZ, CTX and ATZ were used for ESBL. ABPC was employed
for -lactamase-negative ampicillin-resistant (BLNAR) Haemophilus
sp. Bacteria were adjusted with phosphate-buffered saline to a turbidity equal to that of the 0.5 McFarland standard, and then diluted
10-fold. The prepared bacteria suspension was further diluted with
antibiotic solution to provide a nal inoculum density of approximately 10 5 CFU/ml in each well. Each well of a plate was inoculated
with 100 l inoculum, and the plate was incubated for 2496 h at
35 C. After incubation, the MIC was read as the lowest concentration
of antibiotic at which there was no visible growth. In addition, the
turbidity was also measured at 610 nm using a microplate reader
(VersaMax, Molecular Devices Co., Sunnyvale, CA, USA).
For the susceptibility testing using the proposed method, the inoculum (190 l) prepared as described above, was incubated for 22 or
46 h at 35 C, and then 10 l of the detection reagent was added to
each well. After incubation for 2 h at 35 C, the formazan produced
was measured at 460 nm with a microplate reader. The MIC was
read as the lowest concentration of antimicrobial agent at which the
absorbance change was less than 0.05 versus the blank value that
was obtained without bacteria.
3. Results
3.1. Effect of incubation time on the determination of MIC by a broth
microdilution method

2.3. Antibiotics
The representative antibiotics employed in this study are as follows: penicillins (ampicillin (ABPC), oxacillin (MPIPC), penicillin G

To conrm that an extension of incubation time is necessary to obtain consistent MICs for drug-resistant bacteria using a broth microdilution method, the effects of incubation time on the susceptibility

162

T. Tsukatani et al. / Journal of Microbiological Methods 90 (2012) 160166

curves using representative drug-resistant bacteria and reference antibiotics were studied (Fig. 1). The microbial turbidity was measured
sequentially at 610 nm using a microplate reader. The MICs were also
determined visually and then the results agreed with those obtained
from the turbidity obtained from the microplate reader. For the
MRSA, the MIC towards MPIPC was estimated to be 32 g/ml at an incubation time of 24 h. However, the MIC increased with increasing incubation time and was 256 g/ml at 72 h (Fig. 1(A)). Similarly, the
MIC for VCM against VRE was estimated to be 16 g/ml at 24 h,
while the MIC obtained at 72 h increased to 32 g/ml (Fig. 1(B)). In
MDRP, the MIC for CPFX increased from 8 to 32 g/ml with increasing
incubation time (Fig. 1(C)). On the other hand, MICs obtained at 24 h
were almost equal to those obtained at 96 h against ESBL (64 g/ml)
and BLNAR (4 g/ml) (Fig. 1(D) and (E)).
In MRSA, VRE and MDRP, the bacterial growth, which cannot be
detected with a turbidity method in 24 h, can be detected with increasing the incubation time. This is likely because the bacterial
growth is delayed by antibiotics above a certain concentration. The
increase of MICs with extending the incubation time is thought to
be due to the decrease of the inhibition by antibiotics.

The broth microdilution method recommended by CLSI proposes at

least 1620 h of incubation time to obtain the nal antimicrobial susceptibility results (CLSI, 2006). However, it appeared that this incubation
time was too short to determine consistent MICs for several drugresistant bacteria such as MRSA, VRE and MDRP using the CLSI method
as shown in Fig. 1. It has been reported that the CLSI method at an incubation time of 24 h has the possibility to overlook drug-resistant bacteria
(Aldridge et al., 1983; Boyce et al., 1984; Sahmi and Olsen, 1990; Woods
et al., 1986). Thus, a method which provides consistent susceptibility testing for these bacteria would be useful. To evaluate the applicability of the
WST-8 colorimetric method to rapid susceptibility testing, MICs determined by the present method were compared with those obtained by
the broth microdilution method at various incubation times. For the present method, after incubation, the detection reagent was incubated for a
further 2 h with the test substance.
S. aureus, E. faecalis, P. aeruginosa, K. pneumoniae and H. inuenzae
were employed as representative drug-resistant or -susceptible bacteria. Table 1 shows the effects of the incubation time on the

(D) Klebsiella pneumoniae ATCC700603 (ESBL) vs. ceftazidime

(A) Staphylococcus aureus JCM8702 (MRSA) vs. oxacillin

(g/ml)
256
128

0.4

64

0.3

32
16

0.2

8
1

0.1

0.5

(g/ml)

1.2

Absorbance (610nm)

0.5

Absorbance (610nm)

3.2. Determination of consistent MIC in drug-resistant bacteria

256

1.0

128
64

0.8

32
16

0.6

0.4

0.2

2
1

0.25

24

48

72

96

24

0.0625

Time (h)

1024

0.4

512
256

0.3

128
64

0.2

32
16

0.1

72

24

48

72

96

Time (h)

2
1
0

(g/ml)

0.4

128
64

0.3

32
16

0.2

8
4
2

0.1

1
0.5

0.0
0

24

48

72

96

Time (h)

(C) Pseudomonas aeruginosa GTC02017 (MDRP) vs. ciprofloxacin

(g/ml)

Absorbance (610nm)

1.2

64

1.0

32

0.8

16
8

0.6

4
2

0.4

1
0.5

0.2

0.25

0.0

24

48

Time (h)

72

96

0.25
0

0.0

0.5

96

(E) Haemophilus influenzae ATCC49247 (BLNAR) vs. ampicillin

(g/ml)

0.5

48

Time (h)

(B) Enterococcus faecalis GTC02000 (VRE) vs. vancomycin

Absorbance (610nm)

0.0

0.125

Absorbance (610nm)

0.0

0.125
0.0625
0

Fig. 1. Effects of incubation time on susceptibility curves using representative drug-resistant bacteria and reference antibiotics.

0.25
0.125
0

T. Tsukatani et al. / Journal of Microbiological Methods 90 (2012) 160166

163

Table 1
MICs for oxacillin determined by the WST-8 colorimetric method and the broth microdilution method in Staphylococcus aureus.
Bacteria

WST-8 colorimetric method

Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus

ATCC29213
NBRC12732
ATCC33591, MRSA
ATCC43300, MRSA
JCM8702, MRSA

Broth microdilution method

CLSI

Additional incubation

22 h2 h

46 h2 h

24 h

48 h

72 h

96 h

0.5
1
562
16
128256

0.5
1
562
16
128256

0.250.5
0.51
128
8
32

0.5
0.51
256562
816
64128

0.5
0.51
562
816
128256

0.5
0.51
562
16
128256
(g/ml)

determination of MICs for MPIPC against methicillin-resistant and

-susceptible S. aureus. For methicillin-susceptible S. aureus, the MICs
obtained by the broth microdilution method were almost constant regardless of incubation time. However, the MICs increased with increasing incubation time for MRSA. It took at least 72 h to obtain
constant MIC values for MRSA by the broth microdilution method.
For both the methicillin-resistant and -susceptible S. aureus bacteria,
the MICs obtained by the present method were unchanged regardless
of incubation time (total 24 or 48 h). There was good agreement between the MICs obtained at 24 h using the WST-8 colorimetric method and those obtained after 72 or 96 h using the broth microdilution
method. Thus, the CLSI method at 24 h might give lower MICs for
MPIPC in MRSA.
Similarly, the MIC for VCM against VRE using the broth microdilution method increased with increasing incubation time (Table 2).
It took more than 48 h to obtain a consistent MIC value for VRE by the
broth microdilution method. However, for vancomycin-susceptible E.
faecalis, the MICs were almost constant regardless of incubation time
when using the broth microdilution method. The MICs obtained at
24 h using the proposed method agreed with those obtained after
4896 h using the broth microdilution method.
For MDRP, the MICs obtained by the CLSI broth microdilution
method at 24 h were 8, 64, 128 and 16 g/ml for CPFX, ATZ, AMK
and GM, respectively (Table 3(B)). At the incubation time of 24 h,
the MICs determined by the present method were 32, 128256, 256
and 32 g/ml for CPFX, ATZ, AMK and GM, respectively. Lower MICs
were obtained by the CLSI method at 24 h because it is thought that
the incubation period of bacteria and antibiotics might be insufcient
for visual detection of the growth. There was good agreement between the MICs obtained at 24 h using the WST-8 colorimetric method
and those obtained after 4896 h using the broth microdilution
method. For PIPC, CAZ and IPM, the MICs were almost constant regardless of incubation time for either method. On the other hand, in drugsusceptible P. aeruginosa, the MICs were almost constant regardless
of incubation time in the broth microdilution method with the
exception of CPFX and AMK (Table 3(A)). The MICs for CPFX and
AMK towards drug-susceptible P. aeruginosa became constant after
48 h. The antibiotics used in this study are usually employed as
reference antibiotics in antimicrobial susceptibility testing for
MDRP. These ndings indicated that the CLSI method at 24 h tended

to give lower MIC value for some of reference antibiotics towards

MDRP.
Tables 4 and 5 show the effects of the incubation time on the determination of MICs for ESBL and BLNAR, respectively. For both the
drug-resistant and -susceptible bacteria, the MICs obtained by the
broth microdilution method were unchanged regardless of incubation time, and they agreed with those determined by the WST-8 colorimetric method. These results suggested that an incubation time of
24 h was sufcient to determine consistent MICs for several drugresistant bacteria such as ESBL and BLNAR.
For the broth microdilution method, the extension of incubation
time is necessary to obtain consistent MICs in MRSA, VRE and MDRP
(Fig. 1). Therefore, these ndings suggest that the present method
provides a useful means for the rapid determination of consistent
MICs for drug-resistant bacteria, especially MRSA, VRE and MDRP.
3.3. Applications for the determination of consistent MICs in the presence
of various combinations of bacteria and antibiotics
To evaluate the utility of the present method the rapid determination of antimicrobial susceptibility, we used this approach to assess
the susceptibility of quality control strains and/or type strains in the
presence of various antibiotics.
Enterobacteriaceae (E. coli, K. pneumoniae, Salmonella enterica
and Serratia marcescens), P. aeruginosa, A. baumannii, other nonenterobacteriaceae (Alcaligenes faecalis), H. inuenzae, and N.
meningitides were employed as representative gram-negative bacteria. Staphylococcus sp., Enterococcus sp., and Streptococcus sp. were used
as representative gram-positive bacteria. All bacteria used herein are
quality control strains and/or type strains. Penicillins (ABPC, PGC and
MPIPC), cephems (CTX, CAZ and CFIX), penems (IPM), aminoglycosides
(AMK and GM), quinolones (CPFX and LVFX), lipopeptides (CL), macrolides, (CAM), glycopeptides (VCM), phenicols (CP) and tetracyclines
(TC) were applied as the representative antibiotics. Table 6 shows the differences in the MICs determined by the broth microdilution method compared with the present method. At the incubation time of 24 h, there was
58.6% agreement between the MICs obtained by the present method and
the CLSI method. The percentages in MIC values located at 1 and 2
log2 difference were 35.3 and 5.2%, respectively. On the other hand,
there was 92.2% agreement between the MICs obtained by the present

Table 2
MICs for vancomycin determined by the WST-8 colorimetric method and the broth microdilution method in Enterococcus faecalis.
Bacteria

Enterococcus
Enterococcus
Enterococcus
Enterococcus
Enterococcus

WST-8 colorimetric method

faecalis
faecalis
faecalis
faecalis
faecalis

ATCC29212
JCM5803
ATCC51299, VRE
ATCC51299vr, VRE
GTC02000, VRE

Broth microdilution method

CLSI

Additional incubation

22 h2 h

46 h2 h

24 h

48 h

72 h

96 h

4
1
16
128
1632

4
1
16
128
32

4
1
8
3264
16

4
1
16
64
16

4
1
16
64128
1632

4
1
1632
128
32
(g/ml)

164

T. Tsukatani et al. / Journal of Microbiological Methods 90 (2012) 160166

Table 3
MICs for reference antibiotics determined by the WST-8 colorimetric method and the broth microdilution method in Pseudomonas aeruginosa.
Antibiotics

Broth microdilution method

WST-8
colorimetric
method
22 h2 h

(A) Pseudomonas aeruginosa ATCC27853

PIPC
3264
CAZ
16
CPFX
1
ATZ
16
IPM
4
AMK
8
GM
2
(B) Pseudomonas aeruginosa GTC02017, MDRP
PIPC
512
CAZ
64
CPFX
32
ATZ
128256
IPM
32
AMK
256
GM
32

CLSI

Additional incubation

46 h2 h

24 h

48 h

72 h

96 h

3264
1632
1
16
4
8
2

32
16
0.250.5
816
4
24
12

3264
16
0.51
816
4
48
12

3264
16
0.51
16
4
48
12

3264
16
0.51
16
4
48
12

512
64
32
128256
32
256
3264

512
64
8
64
32
128
16

512
64
16
128256
32
128256
1632

512
64
16
128256
32
256
1632

512
64
32
128256
32
256
32
(g/ml)

method at 24 h and the broth microdilution method at 72 h. Furthermore, to better assess the degree of agreement between the MIC results
obtained by the WST-8 colorimetric method and the broth microdilution
method, the Wilcoxon signed-ranked test was performed. P values derived from the Wilcoxon signed-rank test for the incubation time of 24
and 48 h demonstrated signicant differences (Pb 0.001) between both
methods. No difference signicance in the incubation time of 72 h
(P=0.317) emphasized that there was excellent agreement between
the MICs determined by the WST-8 colorimetric method at 24 h and
those obtained by the broth microdilution method at 72 h.
Table 7 shows the list of bacteria that gave higher MICs than the
susceptible breakpoint MICs proposed by CLSI (CLSI, 2008) in
Table 6. The majority of these bacteria had MICs near the breakpoints.
In Enterobacteriaceae such as E. coli, K. pneumoniae, S. enterica and S.
marcescens, the MICs agreed well between the present method and
the broth microdilution method regardless of the incubation time.
On the other hand, for A. baumanni, Enterococcus sp., P. aeruginosa,
and Staphylococcus sp., the MICs obtained by the present method
were higher than those given by the CLSI method at 24 h, and then
agreed with those obtained by the broth microdilution method at
48 or 72 h.
These results suggest that the present method provides a useful
means for the rapid determination of consistent MICs for bacteria
that are known to be resistant to antibiotics near the breakpoint
MICs including staphylococci, A. baumanni, Enterococcus sp., P.

Table 4
MICs for reference antibiotics determined by the WST-8 colorimetric method and the
broth microdilution method in Klebsiella pneumoniae.
Antibiotics

Broth microdilution method

WST-8
colorimetric
method
22 h2 h

46 h2 h

(A) Klebsiella pneumoniae NBRC3512

CAZ
0.25
0.25
CTX
0.015
0.015
ATZ

0.063
0.125
(B) Klebsiella pneumoniae
CAZ
64
CTX
8
ATZ
64128

CLSI

Additional incubation

24 h

48 h

72 h

96 h

0.25
0.007

0.25
0.007
0.015
0.063

0.25
0.007
0.015
0.063

0.25
0.007
0.015
0.063

64
48
64128

64
48
64128

64
48
64128
(g/ml)

0.063
0.063
0.125
ATCC700603, ESBL
64
64
8
4
64128
64128

aeruginosa, and Staphylococcus sp. In addition, it became evident

that the extension of incubation time was necessary to obtain consistent MICs for these bacteria when using the broth microdilution
method.
4. Discussion
The WST-8 colorimetric method for determination of microbial viability was applied to antimicrobial susceptibility testing of various
kinds of bacteria including several drug-resistant types, and its advantages compared to the broth microdilution methods approved
by CLSI were demonstrated. Although the suggested incubation time
to determine the antimicrobial susceptibility of bacteria in the CLSI
method is between 16 and 20 h (CLSI, 2006), it has been reported
that an extension in the incubation time is necessary to increase the
detection efciency of several drug-resistant bacteria in the broth
microdilution method (Aldridge et al., 1983; Boyce et al., 1984;
Hogardt et al., 2004; Sahmi and Olsen, 1990; Woods et al., 1986).
The CLSI method at 24 h has the possibility to overlook drugresistant bacteria. In this study, it was evident that using an incubation time of 24 h in the broth microdilution method gave lower
MICs for MRSA, VRE, and MDRP. This is likely because the incubation
period of bacteria with antibiotics might be insufcient to visually detect changes in growth. The extension of the incubation time to
4896 h was necessary to obtain consistent MICs for these drugresistant bacteria when using the broth microdilution method. CLSI
document M7-A7 details that accurate detection of methicillinresistant staphylococci and VRE requires incubation for a full 24 h
rather than 16 to 20 h (CLSI, 2006). For P. aeruginosa, extended incubation up to 24 h is also recommended in CLSI document M100-S18
(CLSI, 2008). However, this study suggested that even an incubation
Table 5
MICs for ampicillin determined by the WST-8 colorimetric method and the broth microdilution method in Haemophilus inuenzae.
Bacteria

Haemophilus inuenzae
ATCC10211
Haemophilus inuenzae
ATCC49247, BLNAR

Broth microdilution method

WST-8
colorimetric
method

CLSI

Additional incubation

22 h2 h

46 h2 h

24 h

48 h

72 h

96 h

0.25

0.25

0.25

0.25

0.25

0.25

4
(g/ml)

T. Tsukatani et al. / Journal of Microbiological Methods 90 (2012) 160166

165

Table 6
Differences in MICs determined by broth microdilution method at 2472 h compared with the MICs determined by the WST-8 colorimetric method at 24 h.
MIC concordancea

Broth microdilution method

CLSI

Additional incubation

24 h

+2
+1
0
1
2
Total
P
a

48 h

72 h

Number

Number

Number

0
1
68
41
6
116
b 0.001

0.0
0.9
58.6
35.3
5.2
100

0
2
97
16
1
116
b 0.001

0.0
1.7
83.6
13.8
0.9
100

0
3
107
6
0
116
0.317

0.0
2.6
92.2
5.2
0.0
100

Zero indicates number and percentage of strains for which MICs are identical, 2, 1, + 1 and + 2 indicate 2, 1, + 1 and + 2 log2 difference, respectively.
P values were obtained by the Wilcoxon signed-rank test.

time of 24 h might be insufcient for the determination of consistent

MICs for these drug-resistant bacteria. Hopefully, the results from this
study will contribute to the improvement of a standard method for
the determination of antimicrobial susceptibility testing.
Thus, the WST-8 colorimetric method was applied to the antimicrobial susceptibility testing of drug-resistant bacteria such as MRSA, VRE,
and MDRP, and then MICs determined by the present method were
compared with those obtained by the broth microdilution method at
various incubation times. There was excellent agreement between the
MICs determined after 24 h using the WST-8 colorimetric method and
those obtained after 4896 h using the broth microdilution method.
Furthermore, to evaluate the utility of the present method for application in the rapid determination of antimicrobial susceptibility, we
used this approach to assess the susceptibility of various bacteria in
the presence of various antibiotics. The concordance rate of MICs determined by the broth microdilution method to those obtained by the
present method at 24 h increased from 58.6% to 92.2% by the extension
of the incubation time from 24 to 72 h (Table 6). This result suggested
that antimicrobial susceptibility testing could be done more rapidly by
the WST-8 colorimetric method than the broth microdilution method.
Therefore, the present method provides a good alternative for the tedious and time-consuming broth microdilution method.

The list of bacteria shown in Table 7 which gave the higher MICs
than the susceptible breakpoint MICs proposed by CLSI included A.
baumanni and Enterobacteriaceae in addition to Enterococcus sp., P.
aeruginosa, and Staphylococcus sp. The prevalence of clinical infections
caused by multidrug-resistant A. baumanni also continues to increase
throughout the world (Dijkshoorn et al., 2007; Gaynes and Edwards,
2005; Higgins et al., 2010). Furthermore, various emerging infectious
diseases due to multidrug-resistant bacteria such as carbapenemaseproducing K. pneumoniae and gram-negative Enterobacteriaceae with
multidrug-resistance caused by NDM-1 is expanding throughout the
world (Bratu et al., 2005; Kumarasamy et al., 2010; Leavitt et al.,
2007; Nordmann et al., 2009; Nordmann et al., 2011; Yong et al.,
2009). Therefore, the WST-8 colorimetric method would provide a
useful means for rapid determination of antimicrobial susceptibility
testing in emerging infectious diseases.
In the WST-8 colorimetric method, a procedure of the addition of
the detection reagent must be added to the procedure of the broth
microdilution method. However, the proposed method has the advantages as compared to the broth microdilution methods approved
by CLSI. We hope that the WST-8 colorimetric method is used as
one of the alternative methods for the CLSI method for the determination of antimicrobial susceptibility testing.

Table 7
List of the bacteria that gave the higher MICs than the susceptible breakpoint MICs in Table 6.
Bacteria

Escherichia coli NBRC3972

Klebsiella pneumoniae NBRC3512
Salmonella enterica NBRC3313
Serratia marcescens NBRC102204
Acinetobacter baumannii JCM6841

Enterococcus faecalis JCM5803

Enterococcus faecium NBRC100485
Pseudomonas aeruginosa NBRC13275
Staphylococcus aureus NBRC12732
Staphylococcus epidermidis NBRC12993

Antibiotics

CP
ABPC
CP
ABPC
IPM
AMK
CAZ
CPFX
CTX
GM
TC
CPFX
LVFX
CTX
GM
CAZ
CP
CAZ
PCG

WST-8
colorimetric
method

Broth microdilution method

CLSI

Additional incubation

Break point (CLSI)

222 h

24 h

48 h

72 h

16
64
16
64
4
32
32
2
64
3264
8
48
8
32
8
32
16
16
8

16
64
16
64
24
16
16
1
32
32
2
2
4
8
24
16
8
8
4

16
64
16
64
24
32
32
1
3264
32
4
2
8
16
4
32
8
16
8

16
64
16
64
24
32
32
2
3264
64
8
4
8
32
8
32
16
16
8

8
8
8
8
1
16
8
1
8
4
4
1
2
8
4
8
8
8
0.12

16
16
16
16
2
32
16
2
1632
8
8
2
4
1632
8
16
16
16

32
32
32
32
4
64
32
4
64
16
16
4
8
64
16
32
32
32
0.25
(g/ml)

166

T. Tsukatani et al. / Journal of Microbiological Methods 90 (2012) 160166

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