Genetic polymorphism of Aspergillus fumigatus in clinical samples from patients with invasive aspergillosis: investigation using multiple typing methods

The genotypes of 52 strains of Aspergillus fumigatus isolated from 12 patients with invasive aspergillosis were investigated using three typing methods (random amplified polymorphic DNA, sequence-specific DNA polymorphism, and microsatellite polymorphism) combined with multilocus enzyme electrophoresis. Isolates were from patients hospitalized in three different geographic areas (Lyon, France; Grenoble, France; and Milan, Italy). In each case, the genetic polymorphism of several colonies (two to five) within the first respiratory clinical sample was studied. For the 52 isolates tested, random amplified polymorphic DNA identified 8 different genotypes, sequence-specific DNA polymorphism identified 9 different types, and microsatellite polymorphism identified 14 types. A combination of these results with multilocus enzyme electrophoresis study identified 25 different types within the sample studied. We identified 3 patients (of the 12 studied) who carried a single genotype; 6 patients were infected by two genotypes, 1 patient had four genotypes, while the last patient had five. A combination of typing methods provided better discrimination than the use of a single method. Typing methods revealed a population structure within each geographical site, suggesting that the epidemiology of A. fumigatus should be considered separately for each of these geographic areas. This study demonstrates the usefulness of combining several typing methods in reaching an understanding of the epidemiology of A. fumigatus and clarifies whether it is sufficient to type one isolate from each specimen to determine the strain involved in invasive aspergillosis.     

Multicentric epidemiological study of Aspergillus fumigatus isolates by multilocus enzyme electrophoresis.

The genotypes of 63 isolates of Aspergillus fumigatus obtained from three hospitals in different geographical areas and of eight culture collection strains were determined by multilocus enzyme electrophoresis. Twelve of the 17 enzymatic loci studied were polymorphic, giving rise to 48 different electrophoretic types. The existence of fixed multilocus genotypes, significant heterozygote deficits and excesses at the different loci, and linkage disequilibria within subpopulations strongly suggests a clonal reproduction mode for A. fumigatus. Numerical analysis of the comparison and disposition of the different electrophoretic types demonstrates a significant genetic differentiation between the three sampling sites. However, no correlation could be found between geographical distances and genetic differentiation. On account of the multiple discriminatory markers, multilocus enzyme electrophoresis typing seems to be a very powerful tool for epidemiological and reproductive mode studies of A. fumigatus.     

Comparison of multiple typing methods for Aspergillus fumigatus

As part of studies on the spread of infections, risk factors and prevention, several typing methods were developed to investigate the epidemiology of Aspergillus fumigatus . In the present study, 52 clinical isolates of A. fumigatus from 12 airway specimens from patients with invasive aspergillosis (hospitalized in three different centres) were characterized by short tandem repeat (STR) typing and multilocus sequence typing (MLST). These isolates were previously typed by random amplified polymorphic DNA (RAPD), sequence-specific DNA polymorphism (SSDP), microsatellite polymorphism (MSP) and multilocus enzyme electrophoresis (MLEE). STR typing identified 30 genotypes and, for most patients, all isolates were grouped in one cluster of the unweighted pair group method with arithmetic mean dendrogram. Using MLST, 16 genotypes were identified among 50 isolates, while two isolates appeared untypeable. RAPD, MSP, SSDP and MLEE allowed identification of eight, 14, nine and eight genotypes, respectively. Combining the results of these methods led to the delineation of 25 genotypes and a similar clustering pattern as with STR typing. In general, STR typing led to similar results to the previous combination of RAPD, SSDP, MSP and MLEE, but had a higher resolution, whereas MLST was less discriminatory and resulted in a totally different clustering pattern. Therefore, this study suggests the use of STR typing for research concerning the local epidemiology of A. fumigatus , which requires a high discriminatory power.     

Genetic Multilocus Studies of Different Strains of Cryptococcus neoformans: Taxonomy and Genetic Structure

The genotypes of 107 strains of Cryptococcus isolated from the environment or from patients from various geographical areas were determined by multilocus enzyme electrophoresis (MLEE). We analyzed the relationships between genotype structure and serotype and between genotype structure and strain origin. Twelve of the 14 enzyme-encoding loci studied were polymorphic, giving rise to 48 electrophoretic types. The genotypes of C. neoformans and C. laurentii were very similar. MLEE could not distinguish between these two pathogenic species. A correlation between the genetic multilocus structure and the origin of the sample (from the environment or patients) existed. A second analysis detected a correlation between genotype distribution and serotype. The second analysis considered three serotype groups (B, C, and A plus D plus A/D), proving that serotypes A, D, and A/D are closely related. MLEE is a useful epidemiological tool for improving our understanding of the biology of this fungus.     

Comparison of two highly discriminatory molecular fingerprinting assays for analysis of multiple Aspergillus fumigatus isolates from patients with invasive aspergillosis

Two highly discriminatory fingerprinting assays, short tandem repeat typing and amplified fragment length polymorphism (AFLP), were compared to determine the genetic relatedness between 55 isolates of Aspergillus fumigatus obtained from 15 different patients suffering from proven invasive aspergillosis. Both techniques showed that interpatient isolates belonged to different genotypes and that intrapatient isolates from deep sites were all of the same genotype. By contrast, multiple genotypes were found among isolates originating from respiratory samples. Both techniques have specific advantages and disadvantages. AFLP is more universally applicable, but short tandem repeat analysis offers better discriminatory power and should be the preferred method for standardizing typing of clinical isolates of Aspergillus fumigatus.     

Microsatellite analysis of environmental and clinical isolates of the opportunist fungal pathogen Aspergillus fumigatus

Microsatellite analysis was used to examine the genetic relatedness of 111 clinical and environmental isolates of the opportunist human pathogenic fungus Aspergillus fumigatus from Ontario, Canada. Forty-three A. fumigatus isolates were from clinical sources and 68 from environmental sources. Phylogenetic analysis of the genotypes revealed that there were no geographical or temporal associations of clinical or environmental genotypes. In fact, several of the environmental and clinical isolates showed identical (clonal) genotypes from disparate geographical areas. However, a locus by locus examination revealed that there were several significant differences in allele frequencies between clinical and environmental isolates. There may be linkage of certain microsatellite loci with genes affecting virulence in A. fumigatus. A susceptible individual may be equally predisposed to infection by any isolate of A. fumigatus. However, under transient selection as a pathogen, genes encoding alleles for enhanced virulence may not assort independently from microsatellite loci. A dynamic equilibrium may exist between random recombination of loci in the natural environment and selection for virulence factors during host infection cycles.     

Molecular epidemiology of airway colonisation by Aspergillus fumigatus in cystic fibrosis patients

A total of 109 sequential and multiple Aspergillus fumigatus isolates corresponding to 41 samples from seven cystic fibrosis (CF) patients was typed by random amplification of polymorphic DNA (RAPD) with the primer NS3 from the fungal ribosomal gene 18S subunit, and by sequence-specific DNA primer (SSDP) analysis. RAPD typing of the isolates revealed 10 different genotypes, whereas nine genotypes were identified by SSDP. Combination of the two typing methods permitted the differentiation of 25 overall genotypes. The colonisation typing patterns differed greatly between patients colonised for <1 year by A. fumigatus and long-term colonised patients. Two of three recently colonised patients presented a large number of types even in the same sample, unlike the chronically colonised patients, who harboured a limited number of genotypes. In the latter, the occurrence of a dominant genotype, usually the overall genotype 2, tended to reflect to the duration of colonisation. Moreover, anti-catalase antibodies to A. fumigatus appeared in most cases to be in response to genotype 2. These findings suggest that some strains of A. fumigatus may be selected during prolonged colonisation of the airways in CF patients.     

Genotypic characterization of sequential Aspergillus fumigatus isolates from patients with cystic fibrosis.

Twenty-three sequential Aspergillus fumigatus sputum isolates, which had been collected over a period of 2 years, from two patients with cystic fibrosis were genotyped by random amplified polymorphic DNA PCR and restriction fragment length polymorphism analysis. In patient B, one genotype was predominantly present in the sputum samples, while in the other patient up to nine different genotypes were identified. This study suggests that different patterns of colonization with A.fumigatus exist in patients with cystic fibrosis.     

Comparison of three typing methods for clinical and environmental isolates of Aspergillus fumigatus.

To evaluate procedures used for epidemiologic analysis of outbreaks of aspergillosis, we analyzed a collection of 35 Aspergillus fumigatus isolates using three typing methods: isoenzyme analysis (IEA), random amplified polymorphic DNA (RAPD) analysis, and restriction endonuclease analysis (REA). Twenty-one isolates were from a single hospital, with four isolates coming from different patients. Three clinical isolates came from a different hospital, and 11 clinical or environmental isolates were derived from a culture collection. With IEA, the patterns of alkaline phosphatase, esterase, and catalase discriminated nine types. In contrast, 22 types were obtained with five different RAPD primers, and 21 types could be detected with three of these (R108, R151, and UBC90). Restriction endonuclease analysis of genomic DNA, digested with either XbaI, XhoI, or SalI, detected 3, 17, and 13 different REA types, respectively, and 22 types were identified by combining the data from the XhoI and SalI REAs. Twenty-eight types were obtainable with a combination of REA, IEA, and RAPD patterns. Overall, the results pointed to substantial genetic variation among the isolates. Though two isolates had markedly distinct genotypes, their morphologic features and exoantigens were consistent with their being A. fumigatus. The analysis will help in planning epidemiologic studies of aspergillosis.     

Use of variable-number tandem repeats to examine genetic diversity of Neisseria meningitidis

Repetitive DNA motifs with potential variable-number tandem repeats (VNTR) were identified in the genome of Neisseria meningitidis and used to develop a typing method. A total of 146 meningococcal isolates recovered from carriers and patients were studied. These included 82 of the 107 N. meningitidis isolates previously used in the development of multilocus sequence typing (MLST), 45 isolates recovered from different counties in Norway in connection with local outbreaks, and 19 serogroup W135 isolates of sequence type 11 (ST-11), which were recovered in several parts of the world. The latter group comprised isolates related to the Hajj outbreak of 2000 and isolates recovered from outbreaks in Burkina Faso in 2001 and 2002. All isolates had been characterized previously by MLST or multilocus enzyme electrophoresis (MLEE). VNTR analysis showed that meningococcal isolates with similar MLST or MLEE types recovered from epidemiologically linked cases in a defined geographical area often presented similar VNTR patterns while isolates of the same MLST or MLEE types without an obvious epidemiological link showed variable VNTR patterns. Thus, VNTR analysis may be used for fine typing of meningococcal isolates after MLST or MLEE typing. The method might be especially valuable for differentiating among ST-11 strains, as shown by the VNTR analyses of serogroup W135 ST-11 meningococcal isolates recovered since the mid-1990s.     

Molecular typing by random amplification of polymorphic DNA and M13 southern hybridization of related paired isolates of Aspergillus fumigatus.

Three forms of DNA-based typing procedures for Aspergillus fumigatus isolates have been developed over the last five years. The procedures are random amplification of polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) detection, and Southern hybridizations with various repetitive sequence-based probes. Using two of these procedures, we compared 16 selected isolates, grouped into eight pairs on the basis of epidemiology or previously assigned RFLP types. RAPD with four primers (R108, RC08, 2, and 4), including three previously used with A. fumigatus, showed that one primer, R108, gave the best discrimination (8 types). Southern hybridization of total genomic DNA digested with HindIII and probed with the total bacteriophage M13 genome resulted in the highest overall level of discrimination. Combination of the RAPD and Southern hybridization with the previously assigned RFLP types discriminated 10 isolates of 16. Isolates closely linked epidemiologically could not be distinguished from each other. In addition, three pairs of isolates previously unlinked by epidemiology had the same overall types. Two pairs were obtained from the same hospital within 2 years of each other, whereas the third pair were isolated from California and Germany. A full understanding of the epidemiology and ecology of A. fumigatus requires multiple discriminatory typing procedures.     

Differentiation between isolates of Aspergillus fumigatus from breeding turkeys and their environment by genotyping with microsatellite markers

To elucidate the epidemiology of the different forms of avian aspergillosis, 114 Aspergillus fumigatus isolates from sacrificed turkeys and 134 A. fumigatus isolates from air samples were collected and genotyped by microsatellite polymorphism marker analysis. Air sampling confirmed the huge diversity of A. fumigatus populations. Whereas older animals harbored several combinations of genotypes, 1-day-old chicks carried a unique genotype, suggesting a unique source of contamination.     

Immunoblot fingerprinting Aspergillus fumigatus

A new technique for typing Aspergillus fumigatus is presented. This is based on immunoblot fingerprinting each isolate against a rabbit hyperimmune antiserum raised against A. fumigatus NCTC 2109. All isolates were typable and reproducibility for the 16 antigenic bands which formed the basis of the system was excellent. Discrimination was better than silver staining and revealed 11 types among the 21 isolates from eight patients with an aspergilloma. Each aspergilloma could be due to either a single or multiple types.     

Density and Molecular Epidemiology of Aspergillus in Air and Relationship to Outbreaks of Aspergillus Infection

After five patients were diagnosed with nosocomial invasive aspergillosis caused by Aspergillus fumigatus and A. flavus, a 14-month surveillance program for pathogenic and nonpathogenic fungal conidia in the air within and outside the University Hospital in Rotterdam (The Netherlands) was begun. A. fumigatus isolates obtained from the Department of Hematology were studied for genetic relatedness by randomly amplified polymorphic DNA (RAPD) analysis. This was repeated with A. fumigatus isolates contaminating culture media in the microbiology laboratory. The density of the conidia of nonpathogenic fungi in the outside air showed a seasonal variation: higher densities were measured during the summer, while lower densities were determined during the fall and winter. Hardly any variation was found in the numbers of Aspergillus conidia. We found decreasing numbers of conidia when comparing air from outside the hospital to that inside the hospital and when comparing open areas within the hospital to the closed department of hematology. The increase in the number of patients with invasive aspergillosis could not be explained by an increase in the number of Aspergillus conidia in the outside air. The short-term presence of A. flavus can only be explained by the presence of a point source, which was probably patient related. Genotyping A. fumigatus isolates from the department of hematology showed that clonally related isolates were persistently present for more than 1 year. Clinical isolates of A. fumigatus obtained during the outbreak period were different from these persistent clones. A. fumigatus isolates contaminating culture media were all genotypically identical, indicating a causative point source. Knowledge of the epidemiology of Aspergillus species is necessary for the development of strategies to prevent invasive aspergillosis. RAPD fingerprinting of Aspergillus isolates can help to determine the cause of an outbreak of invasive aspergillosis.     

Use of random amplified microsatellites to type isolates from an outbreak of nosocomial aspergillosis in a general medical ward.

Numerous patients were diagnosed with aspergillosis in a nosocomial outbreak caused by Aspergillus fumigatus and Aspergillus flavus. Thirty-three isolates of the former and 28 isolates of the latter were collected from the hospital environment and from the patients and studied for genetic relatedness by random amplified microsatellites (RAMS) analysis, in which two polymorphic regions were tested. Twenty-eight genotypes of A. fumigatus and 23 genotypes of A. flavus were identified. Four patients were infected by two isolates with the same genotype as the environmental isolates. One clinical genotype was shared by three patients and another was shared by two patients. We found that RAMS was useful for fingerprinting Aspergillus spp.     

Limited genetic diversity of Brucella spp

Multilocus enzyme electrophoresis (MLEE) of 99 Brucella isolates, including the type strains from all recognized species, revealed a very limited genetic diversity and supports the proposal of a monospecific genus. In MLEE-derived dendrograms, Brucella abortus and a marine Brucella sp. grouped into a single electrophoretic type related to Brucella neotomae and Brucella ovis. Brucella suis and Brucella canis formed another cluster linked to Brucella melitensis and related to Rhizobium tropici. The Brucella strains tested that were representatives of the six electrophoretic types had the same rRNA gene restriction fragment length polymorphism patterns and identical ribotypes. All 99 isolates had similar chromosome profiles as revealed by the Eckhardt procedure.     

Genetic diversity among clinical isolates of Candida glabrata analyzed by randomly amplified polymorphic DNA and multilocus enzyme electrophoresis analyses

The genetic diversity of 47 clinical and reference strains of Candida glabrata from several geographical origins and diverse clinical disorders, with different antifungal susceptibilities, as well as their genetic relationships were studied through multilocus enzyme electrophoresis (MLEE) and randomly amplified polymorphic DNA (RAPD) techniques. The genetic diversity estimated for 11 MLEE loci measured as average heterozygosity (h) was 0.055. A high level of genetic relatedness among isolates was established by cluster analysis. Forty-nine RAPD markers were analyzed, and the average genetic diversity among isolates, estimated by Shannon's index (Ho), was 0.372. The PhiST values estimated through an analysis of molecular variance to assess genetic differentiation among isolates revealed no genetic differentiation among them. Our results revealed very low genetic diversity among isolates, a lack of differentiation, and no association with their geographic origin and the clinical characteristics.     

Molecular population genetic analysis of Staphylococcus aureus recovered from cows.

Staphylococcus aureus is one of the most common causes of bovine mastitis. To estimate genetic relationships among S. aureus strains recovered from cows, 357 isolates from milk samples from worldwide localities were examined for electrophoretic variation at 13 metabolic-enzyme loci. Thirty-nine electrophoretic types which represented distinctive multilocus enzyme genotypes were identified, and nearly 90% of all isolates were assigned to one of eight clones. Genetic heterogeneity was found among organisms recovered from dairy herds from which multiple isolates were obtained, indicating that the S. aureus population in a single herd can be multiclonal. Although humans and cows shared 7 of the 39 S. aureus clones, each clone was predominantly associated with one of these host species. These results are consistent with the concept of host specialization among S. aureus clones and imply that successful transfer of bacteria between humans and cows is limited.     

Molecular epidemiology of Aspergillus fumigatus isolates recovered from water,air, and patients shows two clusters of genetically distinct strains

There has been an increase in data suggesting that besides air, hospital water is a potential source of transmission of filamentous fungi, and in particular Aspergillus fumigatus. Molecular characterization of environmental and clinical A. fumigatus isolates, collected prospectively during an 18-month period, was performed to establish if waterborne fungi play a role in the pathogenesis of invasive aspergillosis. Isolates recovered from water (n = 54) and air (n = 21) at various locations inside and outside the hospital and from 15 patients (n = 21) with proven, probable, or possible invasive aspergillosis were genotyped by amplified fragment length polymorphism analysis. Based on genomic fingerprints, the environmental A. fumigatus isolates could be grouped into two major clusters primarily containing isolates recovered from either air or water. The genotypic relatedness between clinical and environmental isolates suggests that patients with invasive aspergillosis can be infected by strains originating from water or from air. In addition, 12 clusters with genetically indistinguishable or highly related strains were differentiated, each containing two to three isolates. In two clusters, clinical isolates recovered from patients matched those recovered from water sources, while in another cluster the clinical isolate was indistinguishable from one cultured from air. This observation might open new perspectives in the development of infection control measures to prevent invasive aspergillosis in high-risk patients. The genetic variability found between airborne and waterborne A. fumigatus strains might prove to be a powerful tool in understanding the transmission of invasive aspergillosis and in outbreak control.     

Analysis of Enterococcus faecalis isolates from intercontinental sources by multilocus enzyme electrophoresis and pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) and multilocus enzyme electrophoresis (MLEE) were compared in this study of 65 Enterococcus faecalis isolates recovered over a 20-year period from diverse geographic sources. Clonal relationships recognized by PFGE were also recognized by MLEE; however, MLEE recognized as greater number of isolates as belonging to clonal groups than did PFGE. Both techniques were reproducible and discriminatory, but MLEE more readily recognized relationships among large numbers of isolates. MLEE confirmed the previously reported clonal spread of beta-lactamase-producing E. faecalis to six hospitals in five states. MLEE provided a useful population framework of the E. faecalis isolates in this sample, while PFGE was able to differentiate among isolates within some MLEE clonal groups.