Significant differences in drug susceptibility among species in the Candida parapsilosis group

Candida parapsilosis family has 3 proposed species: C. parapsilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis. C. parapsilosis sensu stricto had significantly higher caspofungin (CAS) and anidulafungin MICs than C. orthopsilosis or C. metapsilosis; C. metapsilosis was least susceptible to fluconazole. C. parapsilosis sensu stricto more frequently displayed (37%) paradoxical growth in CAS (P < or = 0.02). These species susceptibility differences could affect therapeutic choices.     

Screening of strains of the Candida parapsilosis group of the BCCM/IHEM collection by MALDI-TOF MS

One hundred sixty-three strains stored as Candida parapsilosis in the BCCM/IHEM collection were reidentified based on internal transcribed spacer sequencing: 92% were identified as true C. parapsilosis, while 4.3% and 3% belonged to the closely related species C. metapsilosis and C. orthopsilosis, respectively, providing important epidemiologic information. Furthermore, we showed that matrix-assisted laser desorption ionisation time-of-flight mass spectrometry is a fast method that can discriminate between these species.     

Molecular epidemiology and antifungal susceptibility of Candida parapsilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis in Taiwan

Candida parapsilosis was recently reclassified into 3 closely related species, C. parapsilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis. Variation in susceptibility characteristics and prevalence of the 3 genomic species could have therapeutic and epidemiologic implications. The aim of this study is to characterize the genetic and antifungal susceptibility profiles of 97 C. parapsilosis isolates from 71 patients. Among the 71 nonduplicate isolates, 85.9% (61/71) were identified as C. parapsilosis sensu stricto, 5.6% (4/71) as C. metapsilosis, and 8.5% (6/71) as C. orthopsilosis species based on sequences of the internal transcribed spacer (ITS) region. The delineation of these 3 species is concordant with that achieved by pulsed-field gel electrophoresis of BssHII restriction fragments at 75% similarity. Antifungal susceptibility tests showed that most isolates were susceptible to flucytosine, azoles, amphotericin B, and echinocandins, whereas 3 C. metapsilosis isolates from 1 patient showed resistance and susceptible-dose dependence to fluconazole. The C. metapsilosis isolates exhibited significantly higher MIC values to both fluconazole and voriconazole than those of C. parapsilosis sensu stricto and C. orthopsilosis. On the other hand, the C. metapsilosis isolates showed significantly lower MIC values on 24 h to caspofungin than those of C. parapsilosis sensu stricto and C. orthopsilosis. For micafungin, the isolates of C. parapsilosis sensu stricto had significantly higher MIC values on 24 h than those of C. orthopsilosis and C. metapsilosis. Compared to Candida albicans, mutations from proline to alanine were identified on the hot spot 1 of Fks1 in all these C. parapsilosis sensu lato isolates regardless of their MIC levels. Some of the C. orthopsilosis and C. metapsilosis isolates expressed the isoleucine to valine substitution on the hot spot 2 region. However, the amino acid variations in these isolates did not correlate to their MIC values of echinocandin.     

Microsatellite multilocus genotyping clarifies the relationship of Candida parapsilosis strains involved in a neonatal intensive care unit outbreak

Microsatellite typing of 25 Candida parapsilosis isolates from a described outbreak in a neonatal intensive care showed 2 large groups of blood isolates that were related to hand isolates from specific hospital staff, not infant-colonizing isolates. These results demonstrate the power of this typing tool in clarifying epidemiologic associations.     

A simple xylose-based agar medium for the differentiation of Candida dubliniensis and Candida albicans

The utility of xylose-based agar medium for differentiation of Candida dubliniensis from Candida albicans is evaluated. All C. dubliniensis isolates failed to grow on this medium, while C. albicans isolates yielded good growth. This simple in-house medium offers an inexpensive alternative to commercial yeast identification systems for resource poor settings.     

In vitro biofilm characterization and activity of antifungal agents alone and in combination against sessile and planktonic clinical Candida albicans isolates

Thirty clinical isolates of Candida albicans were collected from blood or other sterile site infections. Biofilm dry weight and metabolic activity were measured for each isolate. Planktonic and sessile antifungal susceptibilities of each isolate were determined for amphotericin B deoxycholate, caspofungin, and voriconazole. Sessile susceptibilities were determined for the combination of caspofungin/voriconazole. No significant differences in biofilm dry weight or metabolic activity were found between bloodstream and other invasive isolates. Planktonic MIC90 values and sessile MIC90 (SMIC90) values were 0.25 and 2, 0.06 and >256, and 0.5 and 2 microg/mL for amphotericin, voriconazole, and caspofungin, respectively. The SMIC90 of the combination of caspofungin/voriconazole against sessile isolates was 0.5/2 microg/mL. Therefore, the source of invasive C. albicans clinical isolates did not affect in vitro biofilm formation. Susceptibility to antifungal agents decreased when C. albicans was associated with biofilm, and the combination of caspofungin/voriconazole did not appear to provide enhanced activity compared with caspofungin alone.     

Molecular characterization of fluoroquinolone-resistant Mycobacterium tuberculosis clinical isolates from Shanghai, China

China is one of the countries with the highest prevalence of fluoroquinolone-resistant (FQ(r)) Mycobacterium tuberculosis. Nevertheless, knowledge on the molecular characterization of the FQ(r)M. tuberculosis strains of this region remains very limited. This study was performed to investigate the frequencies and types of mutations present in FQ(r)M. tuberculosis clinical isolates collected in Shanghai, China. A total of 206 FQ(r)M. tuberculosis strains and 21 ofloxacin-sensitive (FQ(s)) M. tuberculosis strains were isolated from patients with pulmonary tuberculosis in Shanghai. The phenotypic drug susceptibilities were determined by the proportion method, and the mutations inside quinolone resistance-determining region (QRDR) of gyrA and gyrB genes were identified by DNA sequence analyses. Among 206 FQ(r)M. tuberculosis strains, 44% (90/206) were multidrug-resistant isolates and 39% (81/206) were extensively drug-resistant isolates. Only 9% (19/206) were monoresistant to ofloxacin. In total, 79.1% (163/206) of FQ(r) isolates harboured mutations in either gyrA or gyrB QRDR. Mutations in gyrA QRDR were found in 75.7% (156/206) of FQ(r) clinical isolates. Among those gyrA mutants, a majority (75.6%) harboured mutations at amino acid position 94, with D94G being the most frequent amino acid substitution. Mutations in gyrA QRDR showed 100% positive predictive value for FQ(r)M. tuberculosis in China. Mutations in gyrB were observed in 15.5% (32/206) of FQ(r) clinical isolates. Ten novel mutations were identified in gyrB. However, most of them also harboured mutations in gyrA, limiting their contribution to FQ(r) resistance in M. tuberculosis. Our findings indicated that, similar to other geographic regions, mutations in gyrA were shown to be the major mechanism of FQ(r) resistance in M. tuberculosis isolates. The mutations in gyrA QRDR can be a good molecular surrogate marker for detecting FQ(r)M. tuberculosis in China.     

Demonstration and utility of clustered pseudohyphae on Gram-stained smears from Candida albicans-positive blood cultures

The presence of clustered and branched pseudohyphae was investigated on Gram-stained smears of 78 consecutive yeast-positive blood cultures. The accuracy of the method was 96.1%, with a positive predictive value of 96.6% for Candida albicans. These findings demonstrate that the presence of clustered and branched pseudohyphae on Gram stain may be used for the rapid and presumptive identification of C. albicans from yeast-positive blood culture bottles.     

Identification,characterization, and biofilm formation of clinical Chryseobacterium gleum isolates

Chryseobacterium gleum is not commonly isolated from clinical source(s). Using 16S rRNA gene sequencing, we identified 15 C. gleum isolates from the Central Region Hospital Alliance, Taiwan, which were all misidentified: 14 as Chryseobacterium indologenes and 1 as Elizabethkingia meningoseptica using the Vitek 2 GN card. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a rapid and clinically applicable method, was evaluated for the identification of C. gleum, and the rate of species or probable species level identification reached 13.3% and 86.6%, respectively. Using pulsed-field gel electrophoresis analysis, all C. gleum isolates from central Taiwan were found to be epidemiologically unrelated. The most prevalent sample was urine (35.7%, 5/14), followed by sputum (28.6%, 4/14), whereas 1 isolate was from an unknown source. All of the isolates were susceptible to minocycline, 93.3% susceptible to trimethoprim/sulfamethoxazole, but were completely or highly resistant to the other drugs examined. Biofilm-forming ability was observed in 40.0% (6/15) isolates using the Luria–Bertani broth. To the best of our knowledge, this is the first focusing on exploring clinical C. gleum isolates.     

Evaluation of the MALDI TOF-MS method for identification of Candida strains isolated from blood cultures

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF-MS) allows rapid and accurate identification of microorganisms. It is being used increasingly and becoming an important tool in clinical laboratories. Phenotypic identification of Candida species remains labor- and time consuming, and the results may sometimes be inconclusive. Rapid and reliable species identification of Candida is essential for antifungal treatment due to species-specific susceptibility patterns. In this study, we evaluated the performance of MALDI TOF-MS for identification of Candida strains. A total of 281 clinical Candida strains isolated from blood cultures were included in this study. Candida species were identified with conventional methods and automated VITEK 2 YST panels as well as with MALDI TOF-MS. Isolates with discrepant results were subjected to DNA sequencing of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2). Ninety-four percent of the isolates were identified correctly by VITEK 2 and MALDI TOF-MS. Altogether, MALDI-TOF MS yielded the correct species identification for 281 (100%) clinical Candida isolates. MALDI-TOF proved to be a rapid and reliable method for identification of Candida strains in the clinical laboratory.     

Virulence gene profiling and molecular characterization of hospital-acquired Staphylococcus aureus isolates associated with bloodstream infection

A better understanding of virulence gene profiling and molecular characterization of Staphylococcus aureus isolates associated with bloodstream infection (BSI) may provide further insights related to clinical outcomes with these infections. We analyzed 89 S. aureus isolates including 37 MRSA isolates (41.6%) recovered from 89 adult patients with BSI from 4 hospitals in Zhejiang province, eastern China. Thirty-five (94.6%) of MRSA isolates and 4 (7.7%) of methicillin-sensitive S. aureus (MSSA) isolates were resistant to multiple antimicrobials. All isolates harbored at least 2 of 22 possible virulence genes, including sdrC (92.1%), icaA (89.9%), hla (80.9%), clf (69.7%), sea (68.5%), sdrD (67.4%), hlb (67.4%), sdrE (65.2%), sei (51.7%), seg (50.6%), and cna (50.6%). Forty-four (49.4%) of all S. aureus BSI isolates, including 23 (62.2%) of MRSA isolates, harbored ≥10 of the virulence genes evaluated in this study. Sixteen (43.2%) MRSA isolates and 5 (9.6%) MSSA isolates harbored the gene encoding Panton–Valentine leukocidin (PVL). Collective genes for pvl, sdrE, sed, seg, and sei among MRSA isolates were significantly more frequent relative to MSSA isolates (P < 0.05). A total of 22 sequence types (STs), including novel ST2184, ST2199, and ST2200, and 33 spa types, including novel spa types t9530 and t9532, were identified among S. aureus BSI isolates, among which ST188 (15.7%) and ST7 (15.7%), and t091 (12.4%) and t189 (12.4%), seldom noted for Chinese isolates previously, were major STs and spa types, respectively. In contrast to previous reports, no predominant clones were found in the present study. Among the MRSA isolates, although ST239-MRSA-SCCmecIII, predominant clone in China, still represented the most common clone, it only accounted for 18.9%. However, ST188-MRSA- SCCmecIV seldom reported before accounted for 10.8%. Among the MSSA isolates, ST7-MSSA represented the most common clone (23.1%), followed by ST188-MSSA and ST630-MSSA (9.6% each). In conclusion, simultaneous carriage of multiple virulence genes and genetically considerable diversity were common among S. aureus BSI isolates. Furthermore, MRSA isolates exhibited more frequent carriage of superantigen genes and pvl relative to MSSA isolates. Taken together, there are distinctive virulence gene profiling and molecular characteristic among S. aureus isolates associated with bloodstream infection in China.     

Distribution of Candida species isolated from blood cultures in hospitals in Osaka,Japan

BackgroundCandida species are clinically important causes of bloodstream infections because their mortality is very high. Given that some species of Candida are azole-resistant, identifying the distributions of Candida species could facilitate the formulation of an appropriate empirical antifungal therapy. It has been shown that the distribution varies depending on the continent, country, city, and hospital. In this paper, we describe the distributions of species in hospitals in northern Osaka, Japan.MethodWe evaluated blood culture results obtained from six tertiary hospitals in the northern Osaka area between 2004 and 2011. We also obtained comorbidity information from the patients' hospital medical records. Kaplan–Meier curves were drawn to compare the risk of death related to the different species.ResultsOf the 165 cases of candidemia confirmed by blood culture, 66% were male and the mean age was 62 years (range = 0–96). Overall, Candida albicans comprised 70 cases (43%), followed by Candida parapsilosis with 36 cases (22%), Candida glabrata with 25 cases (15%), Candida tropicalis with 11 cases (7%), Candida krusei with 10 cases (6%), and other Candida species with 13 cases (8%). C. tropicalis had higher associated mortality than other species, although it was not statistically significant.ConclusionsC. albicans was the most frequently isolated species, but the proportion of non-albicans Candida species was not negligible. The relatively high frequency of non-albicans Candida species distinguished the Japanese distribution from other areas. This characteristic distribution may have important implications when formulating an empirical antifungal therapy for Japanese clinical practice.     

Type distribution of serogroup 6 Streptococcus pneumoniae and molecular epidemiology of newly identified serotypes 6C and 6D in China

The recently determined serotypes 6C and 6D Streptococcus pneumoniae, as well as subtypes 6B-I and 6B-II, were not reported in China. Among the 171 invasive isolates, 19 were identified as serogroup 6. There were equal distribution (42.1%) of 6B-I and 6B-II, 15.8% of 6A and lack of 6C and 6D. Among 1662 noninvasive isolates, 210 were identified as serogroup 6. The rates of types 6A, 6B-I, 6B-II, 6C, and 6D were 42.4%, 21.0%, 29.1%, 4.8%, and 2.9%, respectively. Subtype 6B-II was more resistant to antibiotics than others. The main sequence types (STs) of serotype 6C and 6D isolates were ST2912 and ST982, respectively. These results suggested that all recognized types of serogroup 6 can be found in China and that subtype 6B-II was more drug resistant. The epidemic STs of serotype 6C and 6D did not show genetic association with the STs spreading in other countries.     

Molecular characterization of foodborne-associated Staphylococcus aureus strains isolated in Shijiazhuang,China, from 2010 to 2012

Staphylococcus aureus is an opportunistic pathogen commonly identified from food poisoning–associated foodstuffs. From 1996 to the present, S. aureus isolates have been found to exhibit increasing resistance to antimicrobial drugs. The aim of this study was to assess the molecular epidemiology properties of various S. aureus isolates through molecular typing and to investigate their characterization based on their production of enterotoxins and hemolysins and their resistance to antibiotics. A total of 78 coagulase-positive staphylococcal strains isolated from food or clinical samples were analyzed. Eight VNTR loci were used to genotype the 78 isolates, and this analysis resulted in 39 different multilocus variable number tandem repeat analysis (MLVA) profiles. The isolates recovered from a single outbreak exhibited the same MLVA profile. According to CLSI, 97.4% of the isolates were resistant to penicillin, whereas only 3.8% were methicillin-resistant Staphylococcus aureus strains. Through multiplex PCR, 87.2% of the isolates were shown to be enterotoxigenic (SEs), and the most common genes present were sea, sem, seg, seu, and sek. In conclusion, this study demonstrates the prevalence of staphylococcal enterotoxins, the contents of virulent factors, and the characteristics of β-lactam antibiotic resistance in 78 S. aureus isolates. These findings emphasize the need to prevent the presence of S. aureus strains and SE production in foods. Our results also demonstrate that MLVA is a useful and powerful method for epidemiological studies of S. aureus. In contrast to multilocus sequence typing, the MLVA method is a simpler and more rapid method for epidemiological typing with a higher discriminatory power.     

Molecular epidemiology of methicillin-resistant Staphylococcus aureus in a Spanish hospital over a 4-year period: clonal replacement,decreased antimicrobial resistance,and identification of community-acquired and livestock-associated clones

This study was carried out on 189 methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates collected in a third-level hospital in Valladolid, Spain, between 2005 and 2008 in order to investigate the changes in molecular epidemiology and genetic backgrounds associated with the changes in resistance phenotypes produced over time. The MRSA isolates were classified as belonging to 10 different clones, including the identification of a novel MRSA clone, ST2422-MRSA-IV, belonging to CC121; 1 CA-MRSA strain from a USA300 clone; another from ST97-MRSA-IV, associated with clones adapted to livestock (LA-MRSA); and 2 strains belonging to a new spa type (t10258) related to the ST8-MRSA-IV clone. Sixty-two percent of the strains belonging to Spanish-prevalent MRSA sequence type ST125 harboured composite or multiple SCCmec elements including SCCmec type IV plus ccrA/B4 (ST125-SCCmec IV/VI). In the years studied, it was observed that ST125-SCCmec IV/VI replaced the multiresistant ST228-SCCmec I previously prevalent, and, as a consequence, decreased gentamicin and clindamycin resistance was further observed.     

The use of hybrid phage displaying antigen epitope and recombinant protein in the diagnosis of systemic Candida albicans infection in rabbits and cancer patients

Hsp90 and Sap2 are 2 immunodominant antigens of Candida albicans. Both of them can induce the production of antibody. In this article, systemically infected rabbits were used to study the Hsp90 and Sap2 antibody production. Also, pET28a-Hsp90 protein, pET28a-Sap2 protein, hybrid phage displaying LKVIRK epitope, and hybrid phage displaying VKYTS epitope were used for diagnosis of the antibody in cancer patients. The results showed that the Sap2 antibody appeared earlier than Hsp90 antibody in systemically infected rabbits. Meanwhile, both of the antibodies can perform protection in rabbits. The conclusion is that Sap2 antibody, which appears at early stage in systemic candidiasis, may be better than Hsp90 antibody for the diagnosis of invasive candidiasis. For 141 sera of cancer patients, 52 sera were detected Sap2 antibody and 57 sera were detected Hsp90 antibody. Only 14 sera contained both the 2 antibodies. Although recombinant protein was slightly more sensitive than hybrid phage, there was no significant difference between them. For its easy preparation, less expensive hybrid phage displaying antigen epitope may be a better agent for diagnosis of candidiasis.     

In vitro antifungal susceptibility and molecular identity of 99 clinical isolates of the opportunistic fungal genus Curvularia

The in vitro antifungal susceptibility of a set of 99 clinical isolates of Curvularia was tested against 9 drugs using a reference microdilution method. The isolates had been identified previously to species level by comparing their ITS rDNA and glyceraldehyde-3-phosphate dehydrogenase gene sequences with those of reference strains. We were able to reliably identify 73.2% of the isolates, the most frequent species being Curvularia aeria, Curvularia geniculata/Curvularia senegalensis, Curvularia lunata, Curvularia inaequalis, Curvularia verruculosa, and Curvularia borreriae. Most of these isolates had been recovered from nasal sinus, which is generally considered one of the most frequent sites of infection by these fungi. In addition, at least 3 phylogenetic species that have not yet been formally described were detected. The most active drugs were the echinocandins, amphotericin B, and posaconazole, whereas voriconazole and itraconazole showed poor activity.     

Antimicrobial susceptibility of Enterobacteriaceae, including molecular characterization of extended-spectrum beta-lactamase-producing species, in urinary tract isolates from hospitalized patients in North America and Europe: results from the SMART study 2009-2010

A real-time quantitative polymerase chain reaction (qPCR) was developed for detection and discrimination of Mycobacterium tuberculosis (H37Rv and H37Ra) and M. bovis bacillus Calmette-Guérin (BCG) of the Mycobacterium tuberculosis complex (MTBC) from mycobacterial other than tuberculosis (MOTT). It was based on the melting curve (Tm) analysis of the gyrB gene using SYBR^® Green I detection dye and the LightCycler 1.5 system. The optimal conditions for the assay were 0.25 μmol/L of primers with 3.1 mmol/L of MgCl₂ and 45 cycles of amplification. For M. tuberculosis (H37Rv and H37Ra) and M. bovis BCG of the MTBC, we detected the crossing points (Cp) at cycles of 16.96 ± 0.07, 18.02 ± 0.14, and 18.62 ± 0.09, respectively, while the Tm values were 90.19 ± 0.06 °C, 90.27 ± 0.09 °C, and 89.81 ± 0.04 °C, respectively. The assay was sensitive and rapid with a detection limit of 10 pg of the DNA template within 35 min. In this study, the Tm analysis of the qPCR assay was applied for the detection and discrimination of MTBC from MOTT.     

Screening for amino acid substitutions in the Candida albicans Erg11 protein of azole-susceptible and azole-resistant clinical isolates: new substitutions and a review of the literature

For several years, azole antifungal drugs have been a treatment option for potentially life-threatening Candida infections. However, azole resistance can occur through various mechanisms such as alterations in ERG11, encoding lanosterol 14α-demethylase (CYP51). In this study, we investigated the antifungal susceptibility to fluconazole, itraconazole, and voriconazole of 73 clinical isolates of Candida albicans. Screening for amino acid substitutions in Erg11 was performed on each of the 73 isolates. Twenty isolates displayed a marked decrease in azole susceptibility. Amino acid substitutions were detected in more than two-thirds of the strains. In all, 23 distinct substitutions were identified. Four have not been described previously, among which N136Y and Y447H are suspected to be involved in azole resistance. We suggest that the high genetic polymorphism of ERG11 must be considered in the rationale design of new azole compounds targeting lanosterol 14α-demethylase. A review of all Erg11 amino acid polymorphisms described to date is given.