Examination of the genetic variability among biofilm-forming Candida albicans clinical isolates

Biofilms are microbial communities encased in a self-produced polymeric matrix and represent a common mode of microbial growth. Candida albicans is able to colonize the surface of catheters, prostheses, and epithelia, forming biofilms that are highly resistant to antimicrobial drugs. The objective of this study was the genotypic characterization of biofilm-forming C. albicans clinical isolates using RAPD (Random Amplified Polymorphic DNA). We have studied 25 clinical isolates of C. albicans from oral cavities, blood, skin, nail, stool, oesophagus biopsy and vaginal fluids from patients suffering from candidiasis. For each strain biofilm formation was analysed by measuring the ability to adhere to and grow on polystyrene plastic surfaces using XTT [2,3-bis(2-methoxi-4nitro-5sulfophenil)-2H tetrazolium-5carboxanilide] reduction assay. The similarity coefficients generated by RAPD using four different primers varied from 49 to 91%, indicating a high degree of genetic variability between the clinical isolates. The dendrogram clustered the isolates in four related groups, all groups included strains with very different abilities to form biofilms. The isolates with similar genotypes often showed very different biofilm formation abilities. Strains were grouped into clusters independently of their clinical sources. Our results suggested that a direct correlation does not exist between the biofilm-forming ability of natural populations of C. albicans and the genotype as determined by RAPD.     

Parity among interpretation methods of MLEE patterns and disparity among clustering methods in epidemiological typing of Candida albicans

The typing of C. albicans by MLEE (multilocus enzyme electrophoresis) is dependent on the interpretation of enzyme electrophoretic patterns, and the study of the epidemiological relationships of these yeasts can be conducted by cluster analysis. Therefore, the aims of the present study were to first determine the discriminatory power of genetic interpretation (deduction of the allelic composition of diploid organisms) and numerical interpretation (mere determination of the presence and absence of bands) of MLEE patterns, and then to determine the concordance (Pearson product-moment correlation coefficient) and similarity (Jaccard similarity coefficient) of the groups of strains generated by three cluster analysis models, and the discriminatory power of such models as well [model A: genetic interpretation, genetic distance matrix of Nei (d(ij)) and UPGMA dendrogram; model B: genetic interpretation, Dice similarity matrix (S(D1)) and UPGMA dendrogram; model C: numerical interpretation, Dice similarity matrix (S(D2)) and UPGMA dendrogram]. MLEE was found to be a powerful and reliable tool for the typing of C. albicans due to its high discriminatory power (>0.9). Discriminatory power indicated that numerical interpretation is a method capable of discriminating a greater number of strains (47 versus 43 subtypes), but also pointed to model B as a method capable of providing a greater number of groups, suggesting its use for the typing of C. albicans by MLEE and cluster analysis. Very good agreement was only observed between the elements of the matrices S(D1) and S(D2), but a large majority of the groups generated in the three UPGMA dendrograms showed similarity S(J) between 4.8% and 75%, suggesting disparities in the conclusions obtained by the cluster assays.     

白念珠菌临床分离调查及基因分型研究

目的 了解白念珠菌临床分离情况,并探讨其药敏结果与基因分型的相关性.方法 回顾性分析本院2011年3～11月间临床分离白念珠菌分布及耐药性;随机选取232株,采用PCR方法扩增白念珠菌25S rDNA基因内含子区进行基因分型研究;采用ATB真菌药敏试剂条进行药敏分析;统计分析药敏结果与基因分型的相关性.结果 期间共检出酵母样真菌973例,占病原菌阳性样本数比率为15.7％ (973/6196);其中分离白念珠菌562株,占58％ (562/973),主要分布科室为呼吸科(39.1％)、老年科(13.2％)、ICU(7.7％)、神经内科(7.5％)、免疫科(6.0％)以及其他科室(26.5％);标本类型以下呼吸道为主(81.7％),其次为尿路(9.4％)、血液(1.8％)等.对氟胞嘧啶、两性霉素B、氟康唑、伊曲康唑及伏立康唑的耐药率分别为0.9％、0％、1.4％、1.6％和1.1％.随机选取的232株白念珠菌经PCR方法可分为3型:A型125株,B型96株,C型11株.各型在5种药物的耐药性上并无差异.结论 临床分离酵母样真菌以白念珠菌为主,感染部位以下呼吸道为主;临床分离株对5种抗真菌药物敏感度较高,主要基因型为A和B型,不同基因分型间药敏结果并无统计学差异.     

rDNA-based genotyping of clinical isolates of Candida albicans

The study presents an analysis of the restriction pattern ofrDNA fragments of 95 C. albicans isolates previously classified on the basis of the presence of the intron in rDNA into genotypes A (62 isolates), B (28), and C (5). Most isolates (61) with genotype A were classified as "subtype a" and one as "subtype d" (Karahan and Akar; 2005). No differences were observed in the restriction patterns of the tested genotype B isolates. Similarly, most genotype C strains (4/5) showed the same restriction pattern. The results indicate low subtyping variations of the analyzed isolates, which is in contrast to published data obtained from a Turkish collection of yeasts.     

Analysis of genetic relatedness of Haemophilus influenzae isolates by multilocus sequence typing

The gram-negative bacterium Haemophilus influenzae is a human-restricted commensal of the nasopharynx that can also be associated with disease. The majority of H. influenzae respiratory isolates lack the genes for capsule production and are nontypeable (NTHI). Whereas encapsulated strains are known to belong to serotype-specific phylogenetic groups, the structure of the NTHI population has not been previously described. A total of 656 H. influenzae strains, including 322 NTHI strains, have been typed by multilocus sequence typing and found to have 359 sequence types (ST). We performed maximum-parsimony analysis of the 359 sequences and calculated the majority-rule consensus of 4,545 resulting equally most parsimonious trees. Eleven clades were identified, consisting of six or more ST on a branch that was present in 100% of trees. Two additional clades were defined by branches present in 91% and 82% of trees, respectively. Of these 13 clades, 8 consisted predominantly of NTHI strains, three were serotype specific, and 2 contained distinct NTHI-specific and serotype-specific clusters of strains. Sixty percent of NTHI strains have ST within one of the 13 clades, and eBURST analysis identified an additional phylogenetic group that contained 20% of NTHI strains. There was concordant clustering of certain metabolic reactions and putative virulence loci but not of disease source or geographic origin. We conclude that well-defined phylogenetic groups of NTHI strains exist and that these groups differ in genetic content. These observations will provide a framework for further study of the effect of genetic diversity on the interaction of NTHI with the host.     

Conservation of genetic linkage in nonisogenic isolates of Candida albicans

A number of laboratories are now engaged in the genetic analysis of Candida albicans. This diploid yeast, the major fungal pathogen of humans, is imperfect. Parasexual techniques have been devised for complementation and recombination analysis in this organism. This paper attempts to address the question of the extent to which nonisogenic strains of C. albicans have conserved a common genetic map. This analysis is a prerequisite for the integration of work done in different laboratories and may also provide useful information on the taxonomy of the genus Candida. The paper also reports the analysis of an interspecific hybrid between C. albicans and Candida stellatoidea. The method employed in these studies was the analysis of the mitotic recombination relationships of a group of linked genes and their centromere. Strains carrying linked auxotrophic mutations were fused with isogenic and nonisogenic complementary strains to form tetraploids. The mitotic recombination analyses of these tetraploids suggest that in the isolates studied the genetic map is conserved. A comparison of tetraploid and diploid mitotic recombination analyses is also presented.     

Resistogram typing of Candida albicans isolates from oral and cutaneous sites in irradiated patients

Thirteen resistogram strains of Candida albicans were found among isolates obtained from the mouth and cutaneous sites of irradiation of 27 patients receiving treatment for oral and laryngeal cancer. In all cases the yeast was recovered from the mouth before treatment, but not from the skin site until after treatment had begun. Of the 27 patients, 25 harboured one or more strains with identical resistograms in both sites.     

Resistogram typing of oral Candida albicans isolates from normal subjects in three successive trials

Sixty-six oral strains of Candida albicans, which had been consecutively isolated from 22 normal, young females in three isolation trials at intervals of one to three weeks, were biotyped by their susceptibility to boric acid, cetrimide, silver nitrate, sodium periodate and sodium selenite. The 66 isolates were grouped into 13 resistogram types. An identical biotype strain was found three times and twice in seven and six each of the 22 subjects in the three isolation trials, respectively. In the remaining nine subjects, different strains were found at the three trials. These results suggested that certain strains tended to persist in the oral cavity of the normal subjects although changes in the biotype of oral C. albicans strains occurred to a certain extent.     

Piperazinyl quinolines as chemosensitizers to increase fluconazole susceptibility of Candida albicans clinical isolates

The effectiveness of the potent antifungal drug fluconazole is being compromised by the rise of drug-resistant fungal pathogens. While inhibition of Hsp90 or calcineurin can reverse drug resistance in Candida, such inhibitors also impair the homologous human host protein and fungal-selective chemosensitizers remain rare. The MLPCN library was screened to identify compounds that selectively reverse fluconazole resistance in a Candida albicans clinical isolate, while having no antifungal activity when administered as a single agent. A piperazinyl quinoline was identified as a new small-molecule probe (ML189) satisfying these criteria.     

Proteolytic activity of clinical Candida albicans isolates in relation to genotype and strain source

Proteolytic activity is regarded as one of the most important virulence factors of Candida albicans. Several authors recently demonstrated that some karyotypes and genotypes harbouring a group I self-splicing intron (CaLSU) located in the gene encoding the large rRNA subunit showed a high level of proteinase production. The aim of this study was to investigate the correlation between the level of proteinase production and the presence of the CaLSU intron in C. albicans isolates originating from the blood and respiratory tracts (sputum/pharyngeal swabs) of patients with and without oropharyngeal candidosis. The results revealed statistically significant differences in genotype distribution and the level of proteinase production between the C. albicans isolates obtained from blood and from the respiratory tract. Genotype A, without the intron, was prevalent in all groups of strains and its prevalence was higher among isolates from blood (75%) and from patients with candidosis (80%) compared with strains from colonisation (as opposed to infection) (57.8%). Isolates from blood produced significantly less proteinase than isolates from the respiratory tract (p<0.02), and this difference should be attributed to lower proteinase production of genotypes B and C from blood compared with genotypes B and C from the respiratory tract (p<0.01). The higher proteinase production of genotype B than of genotype A was found among respiratory tract isolates only. The presented data indicate that the association between proteinase production and the CaLSU intron depends on the strains' population. Further study is needed on well-defined groups of clinical isolates to elucidate whether the observed diversity in proteinase production plays a role in the selection of strains inducing bloodstream infections.     

都柏林念珠菌的鉴定及与白念珠菌3种表型鉴别方法的研究

目的 从临床分离的念珠菌中进一步鉴定都柏林念珠菌,并评价3种表型鉴别白念珠菌和都柏林念珠菌的方法.方法 对17株临床分离并初步鉴定的白念珠菌和1株ATCC白念珠菌标准株,采用PHR1同源序列PCR法检测,鉴定出其中的都柏林念珠菌;分别采用45℃生长试验、YEPD(1%酵母浸膏,2%蛋白胨,2%葡萄糖)液基39℃芽管生成试验、Staib琼脂(鸟食琼脂)厚壁孢子形成试验对两种菌的表型特点进行比较.结果 17株临床分离的白念珠菌中有3株鉴定为都柏林念珠菌;45℃时,两种菌在改良沙堡弱琼脂上均无明显生长,YEPD液基中仅有1株白念珠菌生长良好;YEPD液基39℃培养2种菌均无芽管生成;Staib琼脂培养72h,3株都柏林念珠菌中有2株可形成厚壁孢子,而白念珠菌则无,与PHR1同源序列检测结果基本一致.结论 PHR1同源序列检测是鉴别都柏林念珠菌与白念珠菌的可靠方法,Staib琼脂厚壁孢子形成试验有助于鉴别两菌,45℃生长试验和YEPD液基39℃芽管生成试验则不能有效鉴别两菌.     

Epidemiological investigation and typing of Candida glabrata clinical isolates by FTIR spectroscopy

Candida glabrata has emerged as one of the leading agents of fungal infections and strain typing is essential for epidemiological investigation that is generally achieved by molecular techniques. In this work, we studied twenty-nine C. glabrata strains isolated from different patients, using a phenotypic approach based on Fourier Transform Infrared (FTIR) spectroscopy, which has been in a previous study successfully applied as a rapid typing method for Candida albicans. A two-step procedure was used for the analysis. The first step included sixteen strains for the internal validation phase, which aimed at finding the spectral windows that would provide the best differentiation between strains. In this phase, hierarchical cluster analysis (HCA) carried out using three spectral windows (900-1200, 1540-1800, 2800-3000 cm(-1)) allowed to obtain the best classification, where each patient strains could be clustered together. A genotypic technique based on randomly amplified polymorphic DNA-analysis (RAPD) confirmed these results. In a second step, the external validation phase, thirteen other clinical strains of C. glabrata isolated from multiple sites in four ICU patients, were tested by FTIR spectroscopy. The analysis was based on the spectral regions previously found in the first step. HCA classification of the strains gave four groups, one group per patient. These results suggest that no inter-human transmission took place. This study shows the potential of FTIR approach for typing of C. glabrata with several advantages compared to other techniques. FTIR typing is fast, effective, and reagent free. Moreover, it is applicable to all micro-organisms and requires a small quantity of biomass.     

Value of morphotyping for the characterization of Candida albicans clinical isolates

Until recently, morphotyping, a method evaluating fringe and surface characteristics of streak colonies grown on malt agar, has been recommended as a simple and unexpensive typing method for Candida albicans isolates. The discriminatory power and reproducibility of Hunter's modified scheme of Phongpaichit's morphotyping has been evaluated on 28 C. albicans isolates recovered from the oral cavity of asymptomatic human immunodeficiency virus-positive subjects, and compared to two molecular typing methods: randomly amplified polymorphic DNA (RAPD) fingerprinting, and contour clamped homogeneous electric field (CHEF) electrophoretic karyotyping. Morphological features of streak colonies allowed to distinguish 11 different morphotypes while RAPD fingerprinting yielded 25 different patterns and CHEF electrophoresis recognized 9 karyotypes. The discriminatory power calculated with the formula of Hunter and Gaston was 0.780 for morphotyping, 0.984 for RAPD fingerprinting, and 0.630 for karyotyping. Reproducibility was tested using 43 serial isolates from 15 subjects (2 to 6 isolates per subject) and by repeating the test after one year storage of the isolates. While genetic methods generally recognized a single type for all serial isolates from each of the subjects studied, morphotyping detected strain variations in five subjects in the absence of genetic confirmation. Poor reproducibility was demonstrated repeating morphotyping after one year storage of the isolates since differences in at least one character were detected in 92.9% of the strains.     

Epidemiological investigation and typing of Candida glabrata clinical isolates by FTIR spectroscopy

Candida glabrata has emerged as one of the leading agents of fungal infections and strain typing is essential for epidemiological investigation that is generally achieved by molecular techniques. In this work, we studied twenty-nine C. glabrata strains isolated from different patients, using a phenotypic approach based on Fourier Transform Infrared (FTIR) spectroscopy, which has been in a previous study successfully applied as a rapid typing method for Candida albicans. A two-step procedure was used for the analysis. The first step included sixteen strains for the internal validation phase, which aimed at finding the spectral windows that would provide the best differentiation between strains. In this phase, hierarchical cluster analysis (HCA) carried out using three spectral windows (900–1200, 1540–1800, 2800–3000 cm^− 1) allowed to obtain the best classification, where each patient strains could be clustered together. A genotypic technique based on randomly amplified polymorphic DNA-analysis (RAPD) confirmed these results. In a second step, the external validation phase, thirteen other clinical strains of C. glabrata isolated from multiple sites in four ICU patients, were tested by FTIR spectroscopy. The analysis was based on the spectral regions previously found in the first step. HCA classification of the strains gave four groups, one group per patient. These results suggest that no inter-human transmission took place. This study shows the potential of FTIR approach for typing of C. glabrata with several advantages compared to other techniques. FTIR typing is fast, effective, and reagent free. Moreover, it is applicable to all micro-organisms and requires a small quantity of biomass.     

Genotypic subgrouping of clinical isolates of Candida albicans and Candida dubliniensis by 25S intron analysis

AIMS: To determine the frequency, distribution and association of genotypes of Candida albicans and C. dubliniensis in invasive and noninvasive clinical isolates. METHODS: Twenty-one invasive and 18 noninvasive isolates were examined by PCR amplification of a transposable intron region in the 25S rRNA gene. Isolates were genotyped following analysis of the size of resulting DNA amplicons. The isolates could be subdivided into four genotypes (A-D). RESULTS: There was no significant difference between the frequency and genotype distribution of the invasive and noninvasive Candida isolates. IMPACT OF THE STUDY: Therapeutic prophylaxis against candidal infections remains an area of controversy. Any diagnostic markers that reflect the potential of isolates to become invasive should be fully explored, so that more focused antifungal intervention should be targeted at these patients with these potential invasive markers. This study demonstrated that analysis of the transposable intron region in the 25S rRNA gene may be useful in helping to differentiate C. albicans from C. dubliniensis isolates, without the need for sequence analysis, which may not be readily available at primary diagnostic laboratories. However, employment of this genotypic assay is not a suitable locus to determine invasiveness and other more reliable markers of invasiveness should be sought.     

Overcoming fluconazole resistance in Candida albicans clinical isolates with tetracyclic indoles

Continuing efforts to discover novel means of combating fluconazole resistance in Candida albicans have identified an indole derivative that sensitizes strains demonstrating resistance to fluconazole. This tetracycle (3, ML229) does not appear to act through established Hsp90 or calcineurin pathways to chemosensitize C. albicans, as determined in Saccharomyces cerevisiae models, and may be a useful probe to uncover alternative resistance pathways.     

Comparison of three molecular methods for typing Aeromonas popoffii isolates

Three typing methods, restriction fragment length polymorphism (RFLP) of the 16S-23S intergenic spacer region (ISR), PCR amplification of the enterobacterial repetitive intergenic consensus (ERIC) and of the repetitive extragenic palindromic units (REP), were evaluated for typing 26 isolates of Aeromonas popoffii from different geographical origins. When the methods were independently studied, ERIC showed the highest discriminatory power. When the methods were combined, the best combination of two methods was ERIC with REP since strains showed a tendency to cluster according to their geographical origin. However, this tendency was reinforced with the addition of ISR-RFLP. This revised version was published online in June 2006 with corrections to the Cover Date.