Hsiu-Jung Lo

EMERGENCE OF REDUCED SUSCEPTIBILITY AND RESISTANCE TO FLUOROQUINOLONES IN ESCHERICHIA COLI IN TAIWAN AND CONTRIBUTIONS OF DISTINCT SELECTIVE PRESSURES

ABSTRACT A survey of 1,203 Escherichia coli isolates from 44 hospitals in Taiwan revealed that 136 (11.3%) isolates were resistant to fluoroquinolones and that another 261 (21.7%) isolates had reduced susceptibility. Resistance was more common in isolates responsible for hospital-acquired (mostly in intensive care units) infections (17.5%) than in other adult inpatient (11.4%; P = 0.08) and outpatient isolates (11.9%; P > 0.1). Similarly, reduced susceptibility was more common in isolates responsible for hospital-acquired infections (30.9%) than in other adult inpatient (21.0%; P = 0.04) and outpatient (21.4%; P = 0.06) isolates. Isolates from pediatric patients were less likely to be resistant (1.3 versus 12.0%; P < 0.01) but were nearly as likely to have reduced susceptibility (17.7 versus 21.9%;P > 0.1) as nonpediatric isolates. There was an inverse relationship in the proportion of isolates that were resistant versus the proportion that had reduced susceptibility among isolates from individual hospitals (R = 0.031; P < 0.05). In an analysis of isolates from two hospitals, all 9 resistant strains possessed double point mutations ingyrA and all 19 strains with reduced susceptibility strains had single point mutations; no mutations were found among fully susceptible strains. Risk factors for resistance included underlying cancer (odds ratio [OR], 83; 95% confidence interval [CI95], 7.3 to 2,241; P < 0.001), exposure to a quinolone (OR, undefined; P = 0.02), and exposure to a nonquinolone antibiotic (OR, 20; CI95, 2.2 to 482; P < 0.001); underlying cancer was the only independent risk factor (OR, 83; CI95, 8.6 to 807; P < 0.001). There were no significant associations between any of these factors and reduced susceptibility. Whereas acute and chronic quinolone use in cancer patients is a major selective pressure for resistance, other undetermined but distinct selective pressures appear to be more responsible for reduced susceptibility to fluoroquinolones in E. coli.

CaNdt80 Is Involved in Drug Resistance in Candida albicans by Regulating CDR1

ABSTRACT Overexpression of CDR1, an efflux pump, is one of the major mechanisms contributing to drug resistance in Candida albicans. CDR1 p-lacZ was constructed and transformed into a Saccharomyces cerevisiae strain so that the lacZ gene could be used as the reporter to monitor the activity of the CDR1 promoter. Overexpression of CaNDT80, the C. albicans homolog of S. cerevisiae NDT80, increases the β-galactosidase activity of the CDR1 p-lacZ construct in S. cerevisiae. Furthermore, mutations in CaNDT80 abolish the induction of CDR1 expression by antifungal agents in C. albicans. Consistently, the Candt80/Candt80 mutant is also more susceptible to antifungal drugs than the wild-type strain. Thus, the gene for CaNdt80 may be the first gene among the regulatory factors involved in drug resistance in C. albicans whose function has been identified.

Multilocus Sequence Typing for Analyses of Clonality of Candida albicans Strains in Taiwan

ABSTRACT Multilocus sequence typing (MLST) was used to characterize the genetic profiles of 51 Candida albicans isolates collected from 12 hospitals in Taiwan. Among the 51 isolates, 16 were epidemiologically unrelated, 28 were isolates from 11 critically ill, human immunodeficiency virus (HIV)-negative patients, and 7 were long-term serial isolates from 3 HIV-positive patients. Internal regions of seven housekeeping genes were sequenced. A total of 83 polymorphic nucleotide sites were identified. Ten to 20 different genotypes were observed at the different loci, resulting, when combined, in 45 unique genotype combinations or diploid sequence types (DSTs). Thirty (36.1%) of the 83 individual changes were synonymous and 53 (63.9%) were nonsynonymous. Due to the diploid nature of C. albicans, MLST was more discriminatory than the pulsed-field gel electrophoresis-BssHII-restricted fragment method in discriminating epidemiologically related strains. MLST is able to trace the microevolution over time of C. albicans isolates in the same patient. All but one of the DSTs of our Taiwanese strain collections were novel to the internet C. albicans DST database (http://test1.mlst.net/ ). The DSTs of C. albicans in Taiwan were analyzed together with those of the reference strains and of the strains from the United Kingdom and United States by unweighted-pair group method using average linkages and minimum spanning tree. Our result showed that the DNA type of each isolate was patient specific and associated with ABC type and decade of isolation but not associated with mating type, anatomical source of isolation, hospital origin, or fluconazole resistance patterns.

Colonization of Human Immunodeficiency Virus-Infected Outpatients in Taiwan with Candida Species

ABSTRACT To understand the Candida colonization of human immunodeficiency virus (HIV)-infected outpatients in Taiwan, we have conducted a prospective cohort study of Candida colonization and its risk factors at the National Taiwan University Hospital from 1999 to 2002. More than 50% of the patients were colonized with Candida species, and 12% developed symptomatic candidiasis. Patients colonized with fluconazole-resistant strains of Candida species had a higher prevalence of candidiasis than those colonized with susceptible strains. Our analysis found that antibiotic treatment and lower CD4+ counts (<200 cells/mm3) increased the rate of oropharyngeal candidiasis in HIV-infected patients, while antiretroviral therapy protected patients from the development of candidiasis.

Assessment of Candida glabrata Strain Relatedness by Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing

ABSTRACT In this study, 80 Candida glabrata isolates from intensive care unit and human immunodeficiency virus (HIV)-infected patients were typed by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and mating type class determination. Among the 25 patients with multiple isolates, 19 patients (76%) contained multiple isolates exhibiting identical or highly related PFGE and MLST genotypes, which may indicate the maintenance or microvariation of one C. glabrata strain in each patient. However, isolates from six patients (24%) displayed different sequence types, PFGE genotypes, or mating type classes, which may indicate colonization with more than one clone over time or strain replacement. High correlations among PFGE genotypes, sequence types, and mating types were found (P < 0.01). MLST exhibited less discriminatory power than PFGE with BssHII. The genotypes, sequence types, and mating type classes were independent of anatomic sources, drug susceptibility, and HIV infection status.

Microbiological, Epidemiological, and Clinical Characteristics and Outcomes of Patients with Cryptococcosis in Taiwan, 1997–2010

Background Among members of Cryptococcus neoformans- Cryptococcus gattii species complex, C. neoformans is distributed worldwide whereas C. gattii is considered to be more prevalent in the subtropics and tropics including Taiwan. This nationwide study was undertaken to determine the distribution of genotypes, clinical characteristics and outcomes of 219 patients with proven cryptococcosis at 20 hospitals representative of all geographic areas in Taiwan during 1997–2010. Methods and Findings Of 219 isolates analyzed, C. neoformans accounted for 210 isolates (95.9%); nine isolates were C. gattii (4.1%). The predominant genotype was VNI (206 isolates). The other genotypes included VNII (4 isolates), VGI (3 isolates) and VGII (6 isolates). Antifungal minimal inhibition concentrations higher than epidemiologic cutoff values (ECVs) were found in nine VNI isolates (7 for amphotericin B). HIV infection was the most common underlying condition (54/219, 24.6%). Among HIV-negative patients, liver diseases (HBV carrier or cirrhosis) were common (30.2%) and 15.4% did not have any underlying condition. Meningoencephalitis was the most common presentation (58.9%), followed by pulmonary infection (19.6%) and “others” (predominantly cryptococcemia) (18.7%). The independent risk factors for 10-week mortality, by multivariate analysis, were cirrhosis of liver (P = 0.014) and CSF cryptococcal antigen titer ≥512 (P = 0.020). All except one of 54 HIV-infected patients were infected by VNI genotype (98.1%). Of the 13 isolates of genotypes other than VNI, 12 (92.3%) were isolated from HIV-negative patients. HIV-infected patients compared to HIV-negative patients were more likely to have meningoencephalitis and serum cryptococcal antigen ≥1∶512. Patients infected with C. gattii compared to C. neoformans were younger, more likely to have meningoencephalitis (100% vs. 57%), reside in Central Taiwan (56% vs. 31%), and higher 10-week crude mortality (44.4% vs. 22.2%). Conclusions Cryptococcus neoformans in Taiwan, more prevalent than C. gatii, has a predominant VNI genotype. Isolates with antifungal MIC higher than ECVs were rare.

Azole-resistant Aspergillus fumigatus isolates carrying TR34/L98H mutations in Taiwan

Cumulative evidence described the emergence and geographical expansion of azole‐resistant A. fumigatus associated with azole treatment failure. To investigate the status of azole resistance in A. fumigatus in Taiwan, we studied 38 A. fumigatus clinical isolates cultivated from 31 patients at two teaching hospitals from 2011 to 2014. Three isolates obtained from respiratory samples of two azole‐naïve patients with pulmonary aspergillosis were found to display multi‐azole resistance and cross resistance to agricultural azole fungicides, and all carried TR34/L98H mutations in cyp51A gene. The prevalence rates of azole resistance were 7.9% and 6.5% based on isolates and patients respectively. A phylogenetic analysis suggested genetic diversity of the TR34/L98H isolates in Taiwan, including a unique genotype distinct from strains outside Taiwan. The result underlines the emergence of such isolates in Taiwan as well, emphasising the importance of further surveillance for azole‐resistant A. fumigatus and implementation of strategies that prevent fungicide‐driven resistance selection.

The distribution of species and susceptibility of amphotericin B and fluconazole of yeast pathogens isolated from sterile sites in Taiwan

To study the demographic changes of yeasts causing invasive infections in Taiwan, especially with respect to species distribution and antifungal susceptibility, we analyzed isolates obtained from four sterile sites of patients in 19 hospitals in 2002 (155 strains) and again from the same hospitals in 2006 (208 strains). Blood was the most common source of the yeasts, accounting for 73.8% of the total isolates, followed by ascites (21.5%), cerebrospinal fluid (3%), and synovia (1.7%). Candida albicans was the most frequently recovered species (50.1% of the total), followed by Candida tropicalis (20.7%), Candida glabrata (11.6%), Candida parapsilosis (8.5%), Cryptococcus neoformans (3.9%), Candida krusei (0.8%), and nine other species (4.3%). There were one (0.3%) and seven (1.9%) isolates with minimum inhibitory concentrations (MICs) of amphotericin B > or =2 mg/l after 24 h and 48 h incubation, respectively. In addition, there were 15 (4.3%) and 31 (8.6%) isolates with MICs of fluconazole > or =64 mg/l under the same conditions. The MIC(90) value of amphotericin B was 1 mg/l. The MIC(90) values of fluconazole were 4 mg/l after 24 h incubation and 32 mg/l after 48 h incubation. Interestingly, MICs for fluconazole > or =64 mg/l after 24 h were significantly higher for isolates obtained in 2006 than those in 2002 after 24 h (7.1% vs. 0.7%, p =0.009) and 48 h (13.5% vs. 2%, p =0.0003) incubations. The demographic difference between these two surveys is mainly due to one species, C. tropicalis.

Comparison of Human and Soil Candida tropicalis Isolates with Reduced Susceptibility to Fluconazole

Infections caused by treatment-resistant non-albicans Candida species, such as C. tropicalis, has increased, which is an emerging challenge in the management of fungal infections. Genetically related diploid sequence type (DST) strains of C. tropicalis exhibiting reduced susceptibility to fluconazole circulated widely in Taiwan. To identify the potential source of these wildly distributed DST strains, we investigated the possibility of the presence in soil of such C. tropicalis strains by pulsed field gel electrophoresis (PFGE) and DST typing methods. A total of 56 C. tropicalis isolates were recovered from 26 out of 477 soil samples. Among the 18 isolates with reduced susceptibility to fluconazole, 9 belonged to DST149 and 3 belonged to DST140. Both DSTs have been recovered from our previous studies on clinical isolates from the Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) program. Furthermore, these isolates were more resistant to agricultural azoles. We have found genetically related C. tropicalis exhibiting reduced susceptibility to fluconazole from the human hosts and environmental samples. Therefore, to prevent patients from acquiring C. tropicalis with reduced susceptibility to azoles, prudent use of azoles in both clinical and agricultural settings is advocated.

Molecular epidemiology of long-term colonization of Candida albicans strains from HIV-infected patients

Twenty-one Candida albicans isolates from three HIV-infected patients were collected over a period of 3 years and characterized for fluconazole susceptibility, infectivity and genetic relatedness. Fluconazole resistance was found in five isolates, four exhibited dose-dependent susceptibility and the remainder were fully susceptible to this agent. Pulsed-field gel electrophoresis of SfiI restriction digests of the genomic DNA from the isolates revealed that isolates from the same swab specimen were identical despite differences in susceptibility to fluconazole and isolates recovered over time from the three patients retained clonally related DNA fingerprints within each patient. This small-scale study confirms the persistence of oral colonization of C. albicans strains in HIV-infected patients. Clinical data also suggests that the primary infecting strain may become a persistent colonist in the oral cavity once the immune function of the patient has been restored.