Journal of Microbiological Methods 90 (2012) 160–166

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Journal of Microbiological Methods

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Comparison of the WST-8 colorimetric method and the CLSI broth microdilutionmethod 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 Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center, Kurume 839‐0861, Japanb Dojindo Laboratories, Kumamoto 861‐2202, Japanc Laboratory of Food Bioscience, Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Kumamoto 860‐0082, Japan

⁎ Corresponding author at: Biotechnology and Food Rdustrial Technology Center, 1465‐5 Aikawamachi, K+81 942 30 6644; fax: +81 942 30 7244.

E-mail address: tukatani@fitc.pref.fukuoka.jp (T. Tsu

0167-7012/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.mimet.2012.05.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 February 2012Received in revised form 1 May 2012Accepted 4 May 2012Available online 27 May 2012

Keywords:Drug-resistantElectron mediatorMicroorganismNaphthoquinoneSusceptibility testingTetrazolium salt

The minimum inhibitory concentrations (MICs) obtained from the susceptibility testing of various bacteria toantibiotics were determined by a colorimetric microbial viability assay based on reduction of a tetrazoliumsalt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodiumsalt (WST-8)} via 2-methyl-1,4-napthoquinone as an electron mediator and compared with those obtainedby the broth microdilution methods approved by the Clinical and Laboratory Standard Institute (CLSI). Espe-cially 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 suchas methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococi (VRE) and multi-drug resistant Pseudomonas aeruginosa (MDRP) in the broth microdilution method. There was excellentagreement between the MICs determined after 24 h using the WST-8 colorimetric method and thoseobtained after 48–96 h using the brothmicrodilution method. The results suggest that theWST-8 colorimetricassay 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 micro-organisms continues to increase throughout the world although medi-cal and sanitary efforts have advanced. Recently, in addition to well-known drug-resistant microorganisms such as methicillin-resistantStaphylococcus aureus (MRSA) (Hsueh et al., 2004; Moran et al., 2006),vancomycin-resistant enterococi (VRE) (Deshpande et al., 2007), andextended spectrum β-lactamase (ESBL)-producing Enterobacteriaceae(Pitout and Laupland, 2008; Valverde et al., 2004), new infections dueto multidrug-resistant bacteria have been increasing. Infections due tomultidrug-resistant Pseudomonas aeruginosa (MDRP) (Gaynes andEdwards, 2005; Pagani et al., 2005; Sekiguchi et al., 2007), multidrug-resistant Acinetobacter baumannii (Dijkshoorn et al., 2007; Gaynes andEdwards, 2005; Higgins et al., 2010), and carbapenemase-producingKlebsiella pneumoniae (Bratu et al., 2005; Leavitt et al., 2007;Nordmann et al., 2009) have rapidly emerged. Furthermore, gram-negative Enterobacteriaceae with multidrug-resistance caused by NewDelhi metallo-β-lactamase 1 (NDM-1) are potentially a major global

esearch Institute, Fukuoka In-urume 839‐0861, Japan. Tel.:

katani).

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health problem (Kumarasamy et al., 2010; Nordmann et al., 2011;Yong et al., 2009).

Therefore, rapid and accurate antimicrobial susceptibility testingis increasingly important for appropriate patient management andclinical surveillance. In general, when any type of antimicrobial sus-ceptibility testing is performed in the clinical laboratory, a standardmethod approved by the Clinical and Laboratory Standard Institute(CLSI) (CLSI, 2006) is frequently used. In the broth microdilutionmethod recommended by CLSI, the incubation time is set between16 and 20 h for determining the antimicrobial susceptibility for path-ogenic bacteria. However, the incubation time may differ for someproblem microorganisms possessing resistance which is difficult todetect (CLSI, 2006). In this CLSI document, it is indicated that accuratedetection of methicillin-resistant staphylococci and VRE requires in-cubation for a full 24 h rather than 16 to 20 h (CLSI, 2006). It hasbeen reported that the extension of the incubation period to 48 h sig-nificantly increased the detection rate of methicillin-resistant staphy-lococci (Aldridge et al., 1983; Boyce et al., 1984; Woods et al., 1986).The possibility of enhancing the detection of VRE by extending the in-cubation time to 48 h has been also reported (Sahmi and Olsen,1990). These reports suggested that the MICs determined by the mic-rodilution method increased with increasing incubation time forthese drug-resistant bacteria. Thus, extension of the incubation timemight be necessary to obtain the consistent minimum inhibitory con-centration (MIC) for drug-resistant bacteria.

161T. Tsukatani et al. / Journal of Microbiological Methods 90 (2012) 160–166

Methods for rapid susceptibility testing using tetrazolium salts as in-dicator reagents have been developed (Brady et al., 2007; Meletiadis etal., 2001; Moriartya et al., 2005; Tunney et al., 2004). The most com-monly used tetrazolium salt in colorimetric assays for microorganismtesting 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 beeasily quantified colorimetrically (Meletiadis et al., 2001; Paull et al.,1988; Tunney et al., 2004). However, we have reported that XTT is eas-ily reduced by culture media components such as peptones and gly-cated proteins or some antibiotics (Tsukatani et al., 2009). The non-cellular reduction of tetrazolium salts leads to an underestimation ofthe activity of antimicrobial substances. When XTT was employed, thenon-cellular reductions in Mueller–Hinton broth in the absence of mi-croorganisms were marked. Furthermore, the non-cellular reductionof XTTwas promoted by the addition of antibiotics. We have developeda colorimetricmethod 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 viabilityassay (Tsukatani et al., 2008, 2009). Mueller–Hinton broth gave mini-mal rise to the non-cellular reduction of WST-8. Our results indicatedthat WST-8 was superior to XTT in terms of its reactive efficiency withthe electron mediators (reduced form) produced by microorganismsand of its tolerance to medium components. In this method, 2-methyl-1,4-naphthoquinone (NQ) used as an electronmediatorwas re-duced by microorganisms, and WST-8 was then reduced by the pro-duced naphthohydroquinone to its formazan which exhibits amaximum absorbance at 460 nm.

The purpose of this study is to apply theWST-8 colorimetric meth-od to antimicrobial susceptibility testing for various drug-resistantbacteria and to demonstrate the advantages of the present methodas compared to the broth microdilution methods approved by CLSI.The WST-8 colorimetric method would provide a useful means forthe rapid determination of consistent MICs for several drug-resistant 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-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) was a giftfrom Dojindo (Kumamoto, Japan). Cation-adjusted Mueller–Hintonbroth and Haemophilus test medium (HTM) were purchased fromKyokuto Pharmaceutical Industrial Co. (Tokyo, Japan). Tryptic soybroth, yeast extract, and de Man–Rogosa–Sharpe (MRS) media wereobtained from Difco Laboratories (Detroit, MI, USA). All otherchemicalswere of analytical reagent grade andwere usedwithout fur-ther purification.

2.2. Detection reagents

2-Methyl-1,4-NQ as an electron mediator was dissolved in di-methyl sulfoxide (DMSO) at concentrations of 1.0 mM. WST-8 wasdissolved in distilled water at a concentration of 11.1 mM and the so-lution was sterilized by passing it through a cellulose acetate mem-brane filter (pore size, 0.2 μm; diameter, 13 mm). The tetrazoliumsalt solution was mixed with electron mediators at a ratio of 9:1.The prepared detection reagent contained 10 mM WST-8, 0.1 mM 2-methyl-1,4-NQ, and 10% DMSO.

2.3. Antibiotics

The representative antibiotics employed in this study are as fol-lows: penicillins (ampicillin (ABPC), oxacillin (MPIPC), penicillin G

(PCG), piperacillin (PIPC)), cephems (cefixime (CFIX), cefotaxime(CTX), ceftazidime (CAZ)), monobactams (aztreonam (ATZ)), penems(imipenem (IPM)), aminoglycosides (amikacin (AMK), gentamicin(GM)), quinolones (ciprofloxacin (CPFX), levofloxacin (LVFX)),lipopeptides (colistin (CL)), macrolides (clarithromycin (CAM)), gly-copeptides (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 Re-source Center at the National Institute of Technology and Evaluation(NBRC, Chiba, Japan), the Japan Collection of Microorganisms, RIKENBioResource Center (JCM, Tsukuba, Japan), the American Type CultureCollection (ATCC, Rockville MD, USA), and Gifu University (Gifu,Japan). Enterococcus faecalis ATCC51299vr (VRE) was produced fromE. faecalis ATCC51299 by repeated daily passage in MRS containing in-creasing levels of vancomycin up to 128 μg/ml. Batches of medium(5 ml in a glass test tube) were inoculated from fresh culture platesand incubated for 18 h at the optimum temperature (30 or 37 °C).The culture media contained tryptic soy broth plus yeast for all thebacteria except HTM (Mueller–Hinton broth with 0.5% yeast extract,1.5 mg% hemin and 1.5 mg% NAD) was used for Haemophilus sp. andMRS was used for Enterococcus sp.

2.5. Susceptibility testing

Reference MICs were determined by the broth microdilutionmethod currently recommended by CLSI (CLSI, 2006). Serial two-fold dilutions of each antibiotic were prepared in cation-adjustedMueller–Hinton broth or HTM. Cation-adjusted Mueller–Hintonbroth was used for all bacteria with the exception of Haemophilussp., Neisseria sp. and Streptococcus sp. HTM was used for Haemophilussp. For Neisseria sp. and Streptococcus sp., cation-adjusted Mueller–Hinton broth containing 0.3% horse blood was employed. MPIPC andVCM were used as the reference antibiotics for MRSA and VRE, re-spectively. PIPC, CAZ, CPFX, ATZ, IPM, AMK and GM were employedfor MDRP. CAZ, CTX and ATZ were used for ESBL. ABPC was employedfor β-lactamase-negative ampicillin-resistant (BLNAR) Haemophilussp. Bacteria were adjusted with phosphate-buffered saline to a tur-bidity equal to that of the 0.5 McFarland standard, and then diluted10-fold. The prepared bacteria suspension was further diluted withantibiotic solution to provide a final inoculum density of approxi-mately 105 CFU/ml in each well. Each well of a plate was inoculatedwith 100 μl inoculum, and the plate was incubated for 24–96 h at35 °C. After incubation, the MIC was read as the lowest concentrationof antibiotic at which there was no visible growth. In addition, theturbidity 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 inoc-ulum (190 μl) prepared as described above, was incubated for 22 or46 h at 35 °C, and then 10 μl of the detection reagent was added toeach well. After incubation for 2 h at 35 °C, the formazan producedwas measured at 460 nm with a microplate reader. The MIC wasread as the lowest concentration of antimicrobial agent at which theabsorbance change was less than 0.05 versus the blank value thatwas obtained without bacteria.

3. Results

3.1. Effect of incubation time on the determination of MIC by a brothmicrodilution method

To confirm that an extension of incubation time is necessary to ob-tain consistent MICs for drug-resistant bacteria using a broth micro-dilution method, the effects of incubation time on the susceptibility

162 T. Tsukatani et al. / Journal of Microbiological Methods 90 (2012) 160–166

curves using representative drug-resistant bacteria and reference an-tibiotics were studied (Fig. 1). The microbial turbidity was measuredsequentially at 610 nm using a microplate reader. The MICs were alsodetermined visually and then the results agreed with those obtainedfrom the turbidity obtained from the microplate reader. For theMRSA, the MIC towards MPIPC was estimated to be 32 μg/ml at an in-cubation time of 24 h. However, the MIC increased with increasing in-cubation time and was 256 μg/ml at 72 h (Fig. 1(A)). Similarly, theMIC for VCM against VRE was estimated to be 16 μg/ml at 24 h,while the MIC obtained at 72 h increased to 32 μg/ml (Fig. 1(B)). InMDRP, the MIC for CPFX increased from 8 to 32 μg/ml with increasingincubation time (Fig. 1(C)). On the other hand, MICs obtained at 24 hwere almost equal to those obtained at 96 h against ESBL (64 μg/ml)and BLNAR (4 μg/ml) (Fig. 1(D) and (E)).

In MRSA, VRE and MDRP, the bacterial growth, which cannot bedetected with a turbidity method in 24 h, can be detected with in-creasing the incubation time. This is likely because the bacterialgrowth is delayed by antibiotics above a certain concentration. Theincrease of MICs with extending the incubation time is thought tobe due to the decrease of the inhibition by antibiotics.

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

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

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

0.0

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Fig. 1. Effects of incubation time on susceptibility curves using rep

3.2. Determination of consistent MIC in drug-resistant bacteria

The broth microdilution method recommended by CLSI proposes atleast 16–20 h of incubation time to obtain the final antimicrobial suscep-tibility results (CLSI, 2006). However, it appeared that this incubationtime was too short to determine consistent MICs for several drug-resistant bacteria such as MRSA, VRE and MDRP using the CLSI methodas shown in Fig. 1. It has been reported that the CLSImethod at an incuba-tion time of 24 h has the possibility to overlook drug-resistant bacteria(Aldridge et al., 1983; Boyce et al., 1984; Sahmi and Olsen, 1990; Woodset al., 1986). Thus, amethodwhich provides consistent susceptibility test-ing for these bacteria would be useful. To evaluate the applicability of theWST-8 colorimetric method to rapid susceptibility testing, MICs deter-mined by the present method were compared with those obtained bythe broth microdilution method at various incubation times. For the pre-sent method, after incubation, the detection reagent was incubated for afurther 2 h with the test substance.

S. aureus, E. faecalis, P. aeruginosa, K. pneumoniae and H. influenzaewere employed as representative drug-resistant or -susceptible bac-teria. Table 1 shows the effects of the incubation time on the

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

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

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resentative drug-resistant bacteria and reference antibiotics.

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

Bacteria WST-8 colorimetric method Broth microdilution method

CLSI Additional incubation

22 h–2 h 46 h–2 h 24 h 48 h 72 h 96 h

Staphylococcus aureus ATCC29213 0.5 0.5 0.25–0.5 0.5 0.5 0.5Staphylococcus aureus NBRC12732 1 1 0.5–1 0.5–1 0.5–1 0.5–1Staphylococcus aureus ATCC33591, MRSA 562 562 128 256–562 562 562Staphylococcus aureus ATCC43300, MRSA 16 16 8 8–16 8–16 16Staphylococcus aureus JCM8702, MRSA 128–256 128–256 32 64–128 128–256 128–256

(μg/ml)

163T. Tsukatani et al. / Journal of Microbiological Methods 90 (2012) 160–166

determination of MICs for MPIPC against methicillin-resistant and-susceptible S. aureus. For methicillin-susceptible S. aureus, the MICsobtained by the broth microdilution method were almost constant re-gardless of incubation time. However, the MICs increased with in-creasing incubation time for MRSA. It took at least 72 h to obtainconstant 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 regardlessof incubation time (total 24 or 48 h). There was good agreement be-tween the MICs obtained at 24 h using the WST-8 colorimetric meth-od and those obtained after 72 or 96 h using the broth microdilutionmethod. Thus, the CLSI method at 24 h might give lower MICs forMPIPC in MRSA.

Similarly, the MIC for VCM against VRE using the broth micro-dilution method increased with increasing incubation time (Table 2).It took more than 48 h to obtain a consistent MIC value for VRE by thebroth microdilution method. However, for vancomycin-susceptible E.faecalis, the MICs were almost constant regardless of incubation timewhen using the broth microdilution method. The MICs obtained at24 h using the proposed method agreed with those obtained after48–96 h using the broth microdilution method.

For MDRP, the MICs obtained by the CLSI broth microdilutionmethod at 24 h were 8, 64, 128 and 16 μg/ml for CPFX, ATZ, AMKand GM, respectively (Table 3(B)). At the incubation time of 24 h,the MICs determined by the present method were 32, 128–256, 256and 32 μg/ml for CPFX, ATZ, AMK and GM, respectively. Lower MICswere obtained by the CLSI method at 24 h because it is thought thatthe incubation period of bacteria and antibiotics might be insufficientfor visual detection of the growth. There was good agreement be-tween theMICs obtained at 24 h using theWST-8 colorimetricmethodand those obtained after 48–96 h using the broth microdilutionmethod. For PIPC, CAZ and IPM, theMICswere almost constant regard-less of incubation time for either method. On the other hand, in drug-susceptible P. aeruginosa, the MICs were almost constant regardlessof incubation time in the broth microdilution method with theexception of CPFX and AMK (Table 3(A)). The MICs for CPFX andAMK towards drug-susceptible P. aeruginosa became constant after48 h. The antibiotics used in this study are usually employed asreference antibiotics in antimicrobial susceptibility testing forMDRP. These findings indicated that the CLSI method at 24 h tended

Table 2MICs for vancomycin determined by the WST-8 colorimetric method and the broth microd

Bacteria WST-8 colorimetric method

22 h–2 h 46 h–2 h

Enterococcus faecalis ATCC29212 4 4Enterococcus faecalis JCM5803 1 1Enterococcus faecalis ATCC51299, VRE 16 16Enterococcus faecalis ATCC51299vr, VRE 128 128Enterococcus faecalis GTC02000, VRE 16–32 32

to give lower MIC value for some of reference antibiotics towardsMDRP.

Tables 4 and 5 show the effects of the incubation time on the de-termination of MICs for ESBL and BLNAR, respectively. For both thedrug-resistant and -susceptible bacteria, the MICs obtained by thebroth microdilution method were unchanged regardless of incuba-tion time, and they agreedwith those determined by theWST-8 color-imetric method. These results suggested that an incubation time of24 h was sufficient to determine consistent MICs for several drug-resistant bacteria such as ESBL and BLNAR.

For the broth microdilution method, the extension of incubationtime is necessary to obtain consistent MICs in MRSA, VRE and MDRP(Fig. 1). Therefore, these findings suggest that the present methodprovides a useful means for the rapid determination of consistentMICs for drug-resistant bacteria, especially MRSA, VRE and MDRP.

3.3. Applications for the determination of consistent MICs in the presenceof various combinations of bacteria and antibiotics

To evaluate the utility of the present method the rapid determina-tion of antimicrobial susceptibility, we used this approach to assessthe susceptibility of quality control strains and/or type strains in thepresence of various antibiotics.

Enterobacteriaceae (E. coli, K. pneumoniae, Salmonella entericaand Serratia marcescens), P. aeruginosa, A. baumannii, other non-enterobacteriaceae (Alcaligenes faecalis), H. influenzae, and N.meningitides were employed as representative gram-negative bac-teria. Staphylococcus sp., Enterococcus sp., and Streptococcus sp.were usedas representative gram-positive bacteria. All bacteria used herein arequality control strains and/or type strains. Penicillins (ABPC, PGC andMPIPC), cephems (CTX, CAZ and CFIX), penems (IPM), aminoglycosides(AMK and GM), quinolones (CPFX and LVFX), lipopeptides (CL), mac-rolides, (CAM), glycopeptides (VCM), phenicols (CP) and tetracyclines(TC)were applied as the representative antibiotics. Table 6 shows the dif-ferences in theMICs determined by the brothmicrodilutionmethod com-paredwith the presentmethod. At the incubation time of 24 h, therewas58.6% agreement between the MICs obtained by the present method andthe CLSI method. The percentages in MIC values located at −1 and −2log2 difference were 35.3 and 5.2%, respectively. On the other hand,there was 92.2% agreement between the MICs obtained by the present

ilution method in Enterococcus faecalis.

Broth microdilution method

CLSI Additional incubation

24 h 48 h 72 h 96 h

4 4 4 41 1 1 18 16 16 16–3232–64 64 64–128 12816 16 16–32 32

(μg/ml)

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

Antibiotics WST-8colorimetricmethod

Broth microdilution method

CLSI Additional incubation

22 h–2 h 46 h–2 h 24 h 48 h 72 h 96 h

(A) Pseudomonas aeruginosa ATCC27853PIPC 32–64 32–64 32 32–64 32–64 32–64CAZ 16 16–32 16 16 16 16CPFX 1 1 0.25–0.5 0.5–1 0.5–1 0.5–1ATZ 16 16 8–16 8–16 16 16IPM 4 4 4 4 4 4AMK 8 8 2–4 4–8 4–8 4–8GM 2 2 1–2 1–2 1–2 1–2

(B) Pseudomonas aeruginosa GTC02017, MDRPPIPC 512 512 512 512 512 512CAZ 64 64 64 64 64 64CPFX 32 32 8 16 16 32ATZ 128–256 128–256 64 128–256 128–256 128–256IPM 32 32 32 32 32 32AMK 256 256 128 128–256 256 256GM 32 32–64 16 16–32 16–32 32

(μg/ml)

164 T. Tsukatani et al. / Journal of Microbiological Methods 90 (2012) 160–166

method at 24 h and the broth microdilution method at 72 h. Further-more, to better assess the degree of agreement between the MIC resultsobtained by theWST-8 colorimetric method and the brothmicrodilutionmethod, the Wilcoxon signed-ranked test was performed. P values de-rived from the Wilcoxon signed-rank test for the incubation time of 24and 48 h demonstrated significant differences (Pb0.001) between bothmethods. No difference significance in the incubation time of 72 h(P=0.317) emphasized that there was excellent agreement betweenthe MICs determined by the WST-8 colorimetric method at 24 h andthose obtained by the broth microdilution method at 72 h.

Table 7 shows the list of bacteria that gave higher MICs than thesusceptible breakpoint MICs proposed by CLSI (CLSI, 2008) inTable 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 andthe 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 methodwere higher than those given by the CLSI method at 24 h, and thenagreed with those obtained by the broth microdilution method at48 or 72 h.

These results suggest that the present method provides a usefulmeans for the rapid determination of consistent MICs for bacteriathat are known to be resistant to antibiotics near the breakpointMICs including staphylococci, A. baumanni, Enterococcus sp., P.

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

Antibiotics WST-8colorimetricmethod

Broth microdilution method

CLSI Additional incubation

22 h–2 h 46 h–2 h 24 h 48 h 72 h 96 h

(A) Klebsiella pneumoniae NBRC3512CAZ 0.25 0.25 0.25 0.25 0.25 0.25CTX 0.015 0.015 0.007 0.007–

0.0150.007–0.015

0.007–0.015

ATZ 0.063–0.125

0.063–0.125

0.063 0.063 0.063 0.063

(B) Klebsiella pneumoniae ATCC700603, ESBLCAZ 64 64 64 64 64 64CTX 8 8 4 4–8 4–8 4–8ATZ 64–128 64–128 64–128 64–128 64–128 64–128

(μg/ml)

aeruginosa, and Staphylococcus sp. In addition, it became evidentthat the extension of incubation time was necessary to obtain consis-tent MICs for these bacteria when using the broth microdilutionmethod.

4. Discussion

TheWST-8 colorimetric method for determination of microbial vi-ability was applied to antimicrobial susceptibility testing of variouskinds of bacteria including several drug-resistant types, and its ad-vantages compared to the broth microdilution methods approvedby CLSI were demonstrated. Although the suggested incubation timeto determine the antimicrobial susceptibility of bacteria in the CLSImethod is between 16 and 20 h (CLSI, 2006), it has been reportedthat an extension in the incubation time is necessary to increase thedetection efficiency of several drug-resistant bacteria in the brothmicrodilution 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 drug-resistant bacteria. In this study, it was evident that using an incuba-tion time of 24 h in the broth microdilution method gave lowerMICs for MRSA, VRE, and MDRP. This is likely because the incubationperiod of bacteria with antibiotics might be insufficient to visually de-tect changes in growth. The extension of the incubation time to48–96 h was necessary to obtain consistent MICs for these drug-resistant bacteria when using the broth microdilution method. CLSIdocument M7-A7 details that accurate detection of methicillin-resistant staphylococci and VRE requires incubation for a full 24 hrather than 16 to 20 h (CLSI, 2006). For P. aeruginosa, extended incu-bation up to 24 h is also recommended in CLSI document M100-S18(CLSI, 2008). However, this study suggested that even an incubation

Table 5MICs for ampicillin determined by the WST-8 colorimetric method and the broth mic-rodilution method in Haemophilus influenzae.

Bacteria WST-8colorimetricmethod

Broth microdilution method

CLSI Additional incubation

22 h–2 h 46 h–2 h 24 h 48 h 72 h 96 h

Haemophilus influenzaeATCC10211

0.25 0.25 0.25 0.25 0.25 0.25

Haemophilus influenzaeATCC49247, BLNAR

4 4 4 4 4 4

(μg/ml)

Table 6Differences in MICs determined by broth microdilution method at 24–72 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 48 h 72 h

Number % Number % Number %

+2 0 0.0 0 0.0 0 0.0+1 1 0.9 2 1.7 3 2.60 68 58.6 97 83.6 107 92.2−1 41 35.3 16 13.8 6 5.2−2 6 5.2 1 0.9 0 0.0Total 116 100 116 100 116 100P⁎ b0.001 b0.001 0.317

a 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.

165T. Tsukatani et al. / Journal of Microbiological Methods 90 (2012) 160–166

time of 24 h might be insufficient for the determination of consistentMICs for these drug-resistant bacteria. Hopefully, the results from thisstudy will contribute to the improvement of a standard method forthe determination of antimicrobial susceptibility testing.

Thus, theWST-8 colorimetric method was applied to the antimicro-bial susceptibility testing of drug-resistant bacteria such as MRSA, VRE,and MDRP, and then MICs determined by the present method werecompared with those obtained by the broth microdilution method atvarious incubation times. There was excellent agreement between theMICs determined after 24 h using the WST-8 colorimetric method andthose obtained after 48–96 h using the broth microdilution method.

Furthermore, to evaluate the utility of the present method for appli-cation in the rapid determination of antimicrobial susceptibility, weused this approach to assess the susceptibility of various bacteria inthe presence of various antibiotics. The concordance rate of MICs deter-mined by the broth microdilution method to those obtained by thepresent method at 24 h increased from 58.6% to 92.2% by the extensionof the incubation time from 24 to 72 h (Table 6). This result suggestedthat antimicrobial susceptibility testing could be done more rapidly bythe WST-8 colorimetric method than the broth microdilution method.Therefore, the present method provides a good alternative for the te-dious and time-consuming broth microdilution method.

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

Bacteria Antibiotics WST-8colorimetricmethod

22–2 h

Escherichia coli NBRC3972 CP 16Klebsiella pneumoniae NBRC3512 ABPC 64Salmonella enterica NBRC3313 CP 16Serratia marcescens NBRC102204 ABPC 64

IPM 4Acinetobacter baumannii JCM6841 AMK 32

CAZ 32CPFX 2CTX 64GM 32–64TC 8

Enterococcus faecalis JCM5803 CPFX 4–8Enterococcus faecium NBRC100485 LVFX 8Pseudomonas aeruginosa NBRC13275 CTX 32

GM 8Staphylococcus aureus NBRC12732 CAZ 32

CP 16Staphylococcus epidermidis NBRC12993 CAZ 16

PCG 8

The list of bacteria shown in Table 7 which gave the higher MICsthan 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 infectionscaused by multidrug-resistant A. baumanni also continues to increasethroughout the world (Dijkshoorn et al., 2007; Gaynes and Edwards,2005; Higgins et al., 2010). Furthermore, various emerging infectiousdiseases due to multidrug-resistant bacteria such as carbapenemase-producing K. pneumoniae and gram-negative Enterobacteriaceae withmultidrug-resistance caused by NDM-1 is expanding throughout theworld (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 auseful means for rapid determination of antimicrobial susceptibilitytesting in emerging infectious diseases.

In the WST-8 colorimetric method, a procedure of the addition ofthe detection reagent must be added to the procedure of the brothmicrodilution method. However, the proposed method has the ad-vantages as compared to the broth microdilution methods approvedby CLSI. We hope that the WST-8 colorimetric method is used asone of the alternative methods for the CLSI method for the determina-tion of antimicrobial susceptibility testing.

6.

Broth microdilution method Break point (CLSI)

CLSI Additional incubation

24 h 48 h 72 h S I R

16 16 16 ≦8 16 32≦64 64 64 ≦8 16 32≦16 16 16 ≦8 16 32≦64 64 64 ≦8 16 32≦2–4 2–4 2–4 ≦1 2 4≦16 32 32 ≦16 32 64≦16 32 32 ≦8 16 32≦1 1 2 ≦1 2 4≦32 32–64 32–64 ≦8 16–32 64≦32 32 64 ≦4 8 16≦2 4 8 ≦4 8 16≦2 2 4 ≦1 2 4≦4 8 8 ≦2 4 8≦8 16 32 ≦8 16–32 64≦2–4 4 8 ≦4 8 16≦16 32 32 ≦8 16 32≦8 8 16 ≦8 16 32≦8 16 16 ≦8 16 32≦4 8 8 ≦0.12 – 0.25≦

(μg/ml)

166 T. Tsukatani et al. / Journal of Microbiological Methods 90 (2012) 160–166

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