Association between fluconazole susceptibility and genetic relatedness among Candida tropicalis isolates in Taiwan

Association between fluconazole susceptibility and

genetic relatedness among Candida tropicalis

isolates in Taiwan

Jang-Shiun Wang,

Shu-Ying Li,

Yun-Liang Yang,

Hsiao-Hui Chou

and Hsiu-Jung Lo

Correspondence Hsiu-Jung Lo hjlo@nhri.org.tw

1_{Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan,}

Republic of China

2_{Division of Clinical Research, National Health Research Institutes, 35 Keyan Road, Zhunan Town,}

Miaoli County 350, Taiwan, Republic of China

3_{Division of Laboratory Research and Development, Center for Disease Control, Taipei, Taiwan,}

Republic of China

4_{Department of Biological Science and Technology, National Chiao Tung University, Hsinchu,}

Taiwan, Republic of China

Received 6 April 2006 Accepted 2 January 2007

Among the 162 Candida tropicalis isolates collected in the Taiwan Surveillance of Antimicrobial Resistance of Yeasts in 1999, 23 (14.2 %) had fluconazole MICs ¢64 mg l”1, and thus fulfilled the definition of resistance. Random amplified polymorphic DNA assay showed that all 23 fluconazole-resistance C. tropicalis isolates collected from different hospitals around Taiwan were closely related. Two distinct pulsotypes associated with fluconazole susceptibility were identified when these 23 resistant isolates, along with 13 susceptible ones, were analysed by PFGE.

INTRODUCTION

Although Candida albicans is the most frequently isolated

yeast pathogen causing morbidity in seriously

immuno-compromised hosts (Cheng et al., 2004; Pfaller et al., 2000;

Yang et al., 2004), there has been a shift toward the more

treatment-resistant non-albicans Candida species (Sanglard

& Odds, 2002; Walsh et al., 2004; Yang et al., 2004). We

initiated a nationwide surveillance, the Taiwan Surveillance

of Antimicrobial Resistance of Yeasts (TSARY), in 1999 to

investigate the distribution and drug susceptibility of

Candida species (Yang et al., 2003, 2004). A total of 53

among the 632 isolates collected in the TSARY (in 1999) had

fluconazole MICs ¢64 mg l

. Thus, they were considered

as resistant isolates (Clinical and Laboratory Standards

Institute, 1997). Among them, 23 were Candida tropicalis,

collected from 10 of the 22 participating hospitals.

Clinically, the increase in the rate of fluconazole resistance

in C. tropicalis is of considerable importance since this is

one of the most commonly isolated non-albicans Candida

species (Cheng et al., 2004; Pfaller et al., 2000; Yang et al.,

2004, 2005). Information regarding the genetic background

of the resistant and susceptible isolates may provide further

insight into the distribution pattern and origin of the

resistance. In this study, we hoped to determine whether

these resistant isolates of C. tropicalis are genetically related

using two methods, random amplified polymorphic DNA

(RAPD) assay and PFGE analysis.

METHODS

Organisms and media. Yeast isolates were collected from 22 hospitals participating in the TSARY in 1999 (Lo et al., 2001). These hospitals were located around the island and covered all four geographical regions: the North, Middle, South and East districts. Each hospital was asked to submit up to 10 C. albicans and 40 non-albicans Candida species between 15 April and 15 June 1999. Duplicate isolates from the same patients were excluded. All isolates were stored frozen at 270uC in bead-containing Microbank cryovials (Pro-Lab Diagnostics). In total, the susceptibilities to fluconazole of 162 C. tropicalis isolates were determined. Then, the genetic relatedness of all 23 fluconazole-resistant isolates, which were collected from 10 hospitals, along with 13 susceptible ones, were determined. These 10 hospitals included four in the North district, three in the South district, two in the East district and one in the Middle district of Taiwan. The number of resistant isolates from each hospital ranged from one to five. RAPD. RAPD assays were performed according to a modified protocol specifically for C. tropicalis (Roilides et al., 2003). Amplification reactions (50 ml) were performed with the RP02 (59-GCG ATC CCC A-39) primer and in accordance with the manufacturer’s protocol, except that 5 units Taq DNA polymerase (New England Biolabs) and 1 ml genomic DNA were added to the reaction. PCR was performed as follows: 94uC for 5 min; 36 uC for 5 min and 72uC for 5 min for 4 cycles; 94 uC for 1 min, 36 uC for 1 min and 72uC for 2 min for 30 cycles; and a 10 min extension period Abbreviations: RAPD, random amplified polymorphic DNA; TSARY,

Taiwan Surveillance of Antimicrobial Resistance of Yeasts.

Journal of Medical Microbiology (2007), 56, 650–653 DOI 10.1099/jmm.0.46664-0

at 72uC. The RAPD reactions (10 ml) were analysed on a 2 % agarose gel containing 0.56 TBE plus ethidium bromide (0.4 mg l21). PFGE. PFGE analysis was performed as described in our previous report (Chen et al., 2005). The plug slices were placed in 200 ml buffer 3 solution (100 mM NaCl, 50 mM Tris/HCl, 10 mM MgCl2, 1 mM

DTT) (New England BioLabs) and incubated for 1 h at 50uC. Then they were transferred to 200 ml buffer 3 solution containing 4 units BssHII and incubated at 50 uC overnight. Electrophoreses were performed with a Biometra Rotaphor at pulse time 6–50 s, angle 120u, 180 V in 0.8 % agarose gel with 0.56 TBE for 36 h. After the electrophoresis, the gel was stained in ethidium bromide solution (0.4 mg l21) for 15 min and destained in distilled water.

Dendrogram analysis was performed with Bionumerics software, version 3.0 (Applied Maths). The similarity values of the fingerprints were based on the presence or absence of bands between each profile pair compared. The band inclusion window was adjusted by the size reference markers. The position tolerance was set at 1 % and optimization was set at 0 %. The Dice coefficient was used to analyse the similarities (SAB) of the band

patterns. UPGMA was used for the cluster analysis.

RESULTS AND DISCUSSION

First of all, the genetic relatedness of all 23 resistant isolates

was analysed by RAPD assay as described by Roilides et al.

(2003). Interestingly, all the resistant isolates were closely

related (Fig. 1). There are two possible explanations for this

result. One is that all C. tropicalis isolates are genetically

related due to the intrinsic stability of their genomes. The

other is that all resistant ones were related due to clonal

spreading. To distinguish between these two possibilities,

we performed PFGE analysis as described previously (Chen

et al., 2005) to determine the genetic relatedness of the 23

resistant isolates along with 13 susceptible ones.

The result of the PFGE analysis is shown in Fig. 2. All of the

36 tested isolates were grouped into one of two pulsotypes,

which were independent of sources and hospitals, but were

closely associated with the fluconazole susceptibility. In

fact, all the 13 susceptible isolates tested belonged to one

pulsotype, while all the 23 resistant ones belonged to the

other. Among the 13 susceptible isolates, two subgroups

with .80 % relatedness were identified, one with 7 and the

other with 5 isolates. Among the 23 resistant ones, a major

subgroup consisting of 19 (82.6 %) isolates with .80 %

relatedness was also identified.

Molecular epidemiological surveillance of Candida species

isolated from an intensive care unit has been performed

Fig. 1. Dendrogram of the 23 fluconazole-resistant C. tropicalis isolates. Cluster analysis was based on the patterns of the RAPD assay. MIC refers to fluconazole. An MIC of 64 indicates the resistant isolates with MIC ¢64 mg l”1. E, East district; M, Middle district; N, North district; S, South district of Taiwan.

Closely related drug-resistant C. tropicalis

using RAPD analysis (Ergon & Gulay, 2005). There were 20

patterns among the 38 C. albicans isolates tested, suggesting

that the source of C. albicans was mostly endogenous,

consistent with other reports (Chen et al., 2001; Li et al.,

2006). In contrast, there were only 3 genotypes among a set

of 15 C. tropicalis isolates (Ergon & Gulay, 2005).

Further-more, through multilocus sequence typing analysis on C.

tropicalis, a set of clustered flucytosine-resistant isolates has

been reported recently (Tavanti et al., 2005). Interestingly, we

have also found relatively few genotypes among the 36

isolates tested by PFGE analysis. In fact, they can be divided

into only two groups as showed in Fig. 2. Whether this

phenomenon is due to clonal spreading or genome stability

needs further investigation. Though resistant isolates were

related, only one group consisted of more than two isolates

exhibiting .90 % relatedness. Therefore, if those resistant

isolates were clonal, microevolution over time has diluted

their genetic closeness. Alternatively, there was selective

pressure that edited out other genetic patterns.

According to the guidelines of the CLSI (Clinical and

Labo-ratory Standards Institute, 1997), the MICs to fluconazole

Fig. 2. Dendrogram of the 23 resistant and 13 susceptible C. tropicalis isolates. Cluster analysis of the 36 isolates was based on the patterns of BssHII restriction endonuclease analysis of genomic DNA. The long solid horizontal line separates the two major pulsotypes. The long dotted horizontal lines show the boundaries between subpulsotypes with .80 % relatedness. MIC refers to fluconazole. An MIC of 64 indicates the resistant isolates with MICs ¢64 mg l”1_{. E, East district; M, Middle district; N,}

North district; S, South district of Taiwan. J.-S. Wang and others

are defined as the MICs of drugs capable of reducing the

turbidity of cells by .50 % after incubation at 35

C for 48 h.

Isolates with MIC ¢64 mg l

are considered to be

fluconazole resistant. Among the phenomena associated

with resistance, ‘trailing’ describes the reduced but persistent

growth that some isolates exhibit at drug concentrations

above the MIC in broth dilution tests with azole antifungal

agents, such as fluconazole (Lee et al., 2004). Trailing may

interfere with the observation of resistance levels in vivo. The

number of isolates exhibiting trailing with a particular MIC

at 48 h is approximately fourfold higher than at 24 h.

Therefore, the trailing growth can make an isolate that

appears susceptible (MIC ,64 mg l

) after 24 h of

incubation to appear resistant (MIC ¢64 mg l

) at 48 h

(Arthington-Skaggs et al., 2002). Thus, we would also like to

determine whether the 23 fluconazole-resistant isolates

exhibit trailing. Of the 23 isolates with MICs ¢64 mg l

at 48 h, only 5, including YM990236, YM990275,

YM-990576, YM990579 and YM990649, exhibited MICs ¢64

mg l

at 24 h. Nevertheless, the data obtained from in vitro

susceptibility testing are not always correlated with in vivo

outcome. Whether these 23 isolates with MICs ¢64 mg l

will cause treatment failure needs further investigation.

The most important finding from this study was that the

fluconazole-resistant C. tropicalis isolates appeared to be

genetically related. Potentially, this will allow the

develop-ment of easy and even rapid identification methods for

clinical fluconazole-resistant or reduced-susceptibility C.

tropicalis using the genotyping information.

ACKNOWLEDGEMENTS

We would like to thank Pfizer for supplying fluconazole. We thank TSARY participating hospitals for providing clinical isolates and information regarding those isolates. We would like to express additional gratitude to the 10 hospitals providing resistant isolates. They are: Chang Gung Memorial Hospital, Buddhist Tzu-Chi General Hospital, Kaohsiung Medical University Chung-Ho Memorial Hospital, Hsin-Chu Hospital, Department of Health, the Executive Yuan, Linkou, Tainan Municipal Hospital, Hsu Foundation, Lo-Tung Poh Ai Hospital, Mackay Memorial Hospital Taitung Branch, Taipei Municipal Zen Ai Hospital, Veterans General Hospital – Kaohsiung, Veterans General Hospital – Taichung. This work was supported in parts by grants DOH94-DC-1102 from Center for Disease Control, Republic of China, NSC-94-2320-B-400-001 and NSC-94-2320-B-009-001 from the National Science Council, and CL-94-PP-05 from the National Health Research Institutes.

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Closely related drug-resistant C. tropicalis