Expression of CgCDR1, CgCDR2, and CgERG11 in Candida glabrata biofilms formed by bloodstream isolates.

Many studies have shown that Candida biofilms are highly resistant to fluconazole. The mechanisms of acquired fluconazole resistance of Candida glabrata in non-biofilms have been elucidated, but the relevance of them relative to C. glabrata in biofilms are unknown. We examined by real-time PCR the expression of CgCDR1, CgCDR2, and CgERG11 of four bloodstream isolates of C. glabrata during the early (6 h), intermediate (15 h), and mature (48 h) phases of biofilm development. We found high levels of biofilm formation in vitro. At 6 and 15 h, the biofilms exhibited, in comparison to planktonic cells, approximately 1.5- and 3.3-fold upregulation of CgCDR1 and 0.5- and 3.1-fold upregulation of CgCDR2, respectively. However, at 48 h, neither gene was upregulated. In comparison, the expression of CgERG11 did not significantly increase during any of the three phases. Our results indicate a temporary increase in the expression of both CgCDR1 and CgCDR2 during the intermediate phase of C. glabrata biofilm development. In addition, the data collected at 48 h suggest that CgCDR1, CgCDR2, and CgERG11 may not play a role in the azole resistance of C. glabrata in mature biofilms.

A dispermic chimera with mixed field blood group B and mosaic 46,XY/47,XYY karyotype.

Chimerism in humans is a rare phenomenon often initially identified in the resolution of an ABO blood type discrepancy. We report a dispermic chimera who presented with mixed field in his B antigen typing that might have been mistaken for the B3 subtype. The propositus is a healthy Korean male blood donor. Neither his clinical history nor initial molecular investigation of his ABO gene explained his mixed field agglutination with murine anti-B. Chimerism was suspected, and 9 short tandem repeat (STR) loci were analyzed on DNA extracted from blood, buccal swabs, and hair from this donor and on DNA isolated from peripheral blood lymphocytes from his parents. The propositus' red blood cells demonstrated mixed field agglutination with anti-B. Exon 6 and 7 and flanking intronic regions of his ABO gene were sequenced and revealed an O01/O02 genotype. B allele haplotype-specific PCR, along with exon 6 and 7 cloning and sequencing demonstrated a third ABO allele, B101. Four STR loci demonstrated a pattern consistent with a double paternal chromosome contribution in the propositus, thus confirming chimerism. His karyotype revealed a mosaic pattern: 32/50 metaphases were 46,XY and 18/50 metaphases demonstrated 47,XYY.

Changes in karyotype and azole susceptibility of sequential bloodstream isolates from patients with Candida glabrata candidemia.

We examined the changes in genotypes and azole susceptibilities among sequential bloodstream isolates of Candida glabrata during the course of fungemia and the relationship of these changes to antifungal therapy. Forty-one isolates were obtained from 15 patients (9 patients who received antifungal therapy and 6 patients who did not) over periods of up to 36 days. The isolates were analyzed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and tested for antifungal susceptibility to fluconazole, itraconazole, and voriconazole. PFGE typing consisted of electrophoretic karyotyping and restriction endonuclease analysis of genomic DNA by use of NotI (REAG-N). The 41 isolates yielded 23 different karyotypes and 11 different REAG-N patterns but only 3 MLST types. The sequential strains from each patient had identical or similar REAG-N patterns. However, they had two or three different karyotypes in 6 (40%) of 15 patients. The isolates from these six patients exhibited the same or similar azole susceptibilities, and five patients did not receive antifungal therapy. Development of acquired azole resistance in sequential isolates was detected for only one patient. For this patient, an isolate of the same genotype obtained after azole therapy showed three- or fourfold increases in the MICs of all three azole antifungals and exhibited increased expression of the CgCDR1 efflux pump. This study shows that karyotypic changes can develop rapidly among sequential bloodstream strains of C. glabrata from the same patient without antifungal therapy. In addition, we confirmed that C. glabrata could acquire azole resistance during the course of fungemia in association with azole therapy.

[Resolution of ABO Discrepancies by ABO Genotyping.].

BACKGROUND: Before a blood transfusion, both red cell and serum typing need to be matched for ABO tests on the donor and patient (recipient). When a mismatch exists in the tests, additional ABO genotyping and serological tests are required for the resolution of the discrepancy. We performed ABO genotyping on a series of blood donors and patients with ABO discrepancies to assist in resolving their blood groups. METHODS: We examined 46 samples with ABO discrepancies from a random pool of donors recruited at Gwangju-Chonnam Red Cross Blood Center and from patients at Chonnam National University Hospital between May 2004 and July 2005. ABO genotyping was performed on all samples with an allele specific polymerase chain reaction for differentiation of A, B,O, cis-AB, A(var) (784 G>A), and B(var) (547 G>A) alleles; routine serologic tests were also performed. Exon 6 and 7 of ABO gene from five samples were sequenced. RESULTS: The genotypes of 18 donors/patients with weakened A or B antigen expressions consisted of 4 cases of cis-AB/O (3 A(2)B(3), 1 A(2)B); 5 cases of cis-AB/A (5 A(1)B(x or el)); 2 cases of A/O (1 O, 1 A(m or x)); 1 case of B/O (1 B(m or x)); 4 cases of A/B (1 A(2)B , 1 A(1)B(x or el), 2 A(1)B(3)); and 2 cases of A(var)/B (2 A(w)B). On the other hand, the genotypes of 28 samples with unexpected serum reactions included 18 cases of A/O (16 A(1), 2 A(int)); 7 cases of A/A (5 A(1), 1 A(1)B(x or el), 1 A(1)B(w)); and 3 cases of O/O (1 O, 2 B(w)). CONCLUSIONS: ABO genotyping is useful for differentiating the ABO discrepancies that were difficult to resolve by serological tests. The most frequent unusual red cell reactions were weak A and B antigen expressions, which were resulted from the ABO subgroup alleles including cis-AB allele, whereas the most frequent unusual serum reactions were caused by decreased anti-B titers.

Differences in biofilm production by three genotypes of Candida parapsilosis from clinical sources.

Three distinct genotypes of Candida parapsilosis (group I, II, and III) have been identified among clinical isolates but their clinical significance remains unclear. We investigated the distribution of C. parapsilosis genotypes in isolates from blood, all other sites from patients, and the hands of health care workers (HCWs), and we examined the relationship between genotype and biofilm positivity. The 53 bloodstream isolates and 38 of 39 HCW isolates were categorized as group I, whereas the 67 non-blood isolates taken from patients were distributed in groups I (n=43), II (n=13), and III (n=11). Biofilm positivity was observed in 77% (103 of 134) of group I isolates versus 0% (0 of 25) of non-group I (groups II and III) isolates (P < 0.01). There was no difference in biofilm production among group I isolates from blood (81%), other clinical specimens (72%), and the hands of HCWs (73%). This study has shown that biofilm production differs among three genotypes of C. parapsilosis isolates and that a majority of C. parapsilosis isolates from the bloodstream (100%), the hands of HCWs (97%), and all other sites from patients (64%) belong to group I, which has the ability to produce biofilm.

Molecular investigation of two consecutive nosocomial clusters of Candida tropicalis candiduria using pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) typing was applied to the epidemiological investigation of 21 Candida tropicalis isolates collected from urine specimens of 11 patients and one healthcare worker, in an intensive care unit (ICU) over a 4-month period. Seventeen epidemiologically unrelated strains from 14 patients were also tested to determine the discriminatory power of PFGE. PFGE typing consisted of electrophoretic karyotyping (EK) and restriction endonuclease analysis of genomic DNA (REAG), using two restriction enzymes (BssHII and SfiI). The EK pattern was the same in all 38 isolates, while REAG using SfiI separated the isolates into nine types. However, 16 different PFGE types were identified by REAG with BssHII, and the same results were obtained when the results of both REAG tests were combined. In serial urinary isolates from 10 patients, all strains from each patient had the same PFGE pattern. While the epidemiologically unrelated strains from 14 patients consisted of 13 different PFGE types, the 20 isolates from the 11 ICU patients fell into only two PFGE types (types C1 and C2), and these apparently originated from the two different outbreaks. All strains of type C1 (n = 12) were isolated from six patients, between November 1999 and January 2000, and all of the type C2 strains (n=8) were isolated from five patients, during January and February 2000. This study shows two consecutive clusters of C. tropicalis candiduria in an ICU, defined by PFGE typing, and also demonstrates that a PFGE typing method using BssHII is perhaps the most useful method for investigating C. tropicalis candiduria.

Microevolution of Candida albicans strains during catheter-related candidemia.

We examined microevolution in a series of Candida albicans strains isolated from patients with catheter-related candidemia. Sixty-one isolates (29 from blood, 18 from catheters, 10 from urine, and 4 from other sites) were obtained from 15 patients who were admitted to the same hospital over a 3-year period. Isolates were analyzed by using Southern hybridization with the C1 fragment of Ca3 as a probe (C1 fingerprinting) and pulsed-field gel electrophoresis (PFGE). PFGE typing consisted of electrophoretic karyotyping (EK) and restriction endonuclease analysis of genomic DNA (REAG) by using SfiI (REAG-S) and BssHII (REAG-B). When catheter isolates were compared with blood isolates from the same patient, catheter isolates from 5 of 14 patients (36%) exhibited minor band differences (microevolution) relative to blood isolates in either C1 fingerprinting (n = 4), REAG-S (n = 3), or REAG-B (n = 5) profiles, although they had identical EK patterns. However, the other sequential isolates from each patient, which had identical EK patterns, showed the same REAG and C1 fingerprinting patterns. Both fingerprinting methods revealed that two distinct genotypes were shared by isolates from seven patients in a neonatal intensive care unit, suggesting two nosocomial clusters. Except for two catheter isolates from the index patients of each cluster, no consecutive isolates collected from each of the two clusters showed any microevolution during the 2- or 7-month cluster periods. The findings suggest that in catheter-related candidemia, some C. albicans strains undergo microevolution during catheter colonization.