Comparison of restriction enzyme analysis and pulsed-field gradient gel electrophoresis as typing systems for Candida albicans.

Candida species are an important cause of infection in immunocompromised hosts and the leading cause of nosocomial fungal infections. Study of the epidemiology of Candida infection has been difficult because of lack of a reliable typing system. We describe a typing system utilizing contour-clamped homogeneous electric fields (CHEF), which is a modified version of pulsed-field gradient gel electrophoresis, and compared it with restriction enzyme analysis (REA) of genomic DNA. The study was done with 35 Candida albicans clinical isolates from separate patients. CHEF and REA were performed on each isolate, and the patterns were compared. The REA procedure revealed 17 strain types while the CHEF procedure was able to distinguish 23 strain types of C. albicans. The CHEF technique yields unique patterns of chromosomal bands that can be used to distinguish clinical isolates and demonstrates greater sensitivity than REA.     

Optimisation of Candida albicans typing by pulsed-field gel electrophoresis.

Six strains of Candida albicans were subjected to pulsed-field gel electrophoresis (PFGE) using the CHEF-DRIII system (BioRad). Hansenula mingei YB-4662-VIA and Saccharomyces cerevisiae YNN 295 (BioRad) were used as size markers (1.05-3.13 and 0.22-2.2 megabase pairs [Mbp] respectively) for comparison of DNA molecules. The DNAs were resolved by a three-block protocol with pulse times of 120 s for 24 h, 240 s for 36 h and 300 s for 17 h. The voltage was set at 4.5 V/cm for the first two blocks and 4.0 V/cm for the final block. PFGE was carried out under these conditions using different agarose concentrations, types and concentrations of buffer, temperatures, and sizes of agarose gel plug. The resolution and mobility of DNAs were affected by some of these variables. Separation of C. albicans by PFGE was optimal at 12 degrees C with 1.0 x Tris-borate-EDTA (TBE) buffer using 1.2% agarose. Resolution of banding patterns was dependent on size of DNA plug used.     

Assessment of Candida glabrata strain relatedness by pulsed-field gel electrophoresis and multilocus sequence typing

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.     

Evaluation of pulsed-field gel electrophoresis as a typing system for Candida rugosa: comparison of karyotype and restriction fragment length polymorphisms.

Nosocomial infections with Candida species have emerged as an increasingly important cause of morbidity and mortality in intensive care units. Ten Candida rugosa isolates from a previously documented cluster of C. rugosa infections in one hospital (nine burn unit isolates and one isolate from another hospital ward) and eight C. rugosa isolates recovered in a referral fungus testing laboratory (comparison isolates) from distinct geographic areas were investigated by molecular techniques. Isolates were from multiple anatomic sites. Pulsed-field gel electrophoresis (PFGE) of whole-cell DNA was performed with the 18 C. rugosa isolates as a marker of strain identity. The PFGE karyotypes of the C. rugosa isolates were demonstrated from four to seven chromosome bands. Karyotyping revealed the same PFGE pattern for the nine outbreak isolates from the burn unit, confirming clonal strain transmission. The isolate from the other hospital ward had a distinct karyotype. Distinct PFGE karyotype patterns were demonstrated for the eight comparison isolates. Restriction fragment length polymorphisms (RFLP) generated from whole-cell DNA digested with SfiI demonstrated the same RFLP pattern among outbreak isolates. Among comparison isolates, karyotyping distinguished some isolates that were indistinguishable by RFLP patterns. Karyotyping by PFGE appears to be the most useful molecular typing tool for discrimination among strains of C. rugosa and will be a useful marker for evaluating the epidemiology of future C. rugosa infections.     

Microsatellite marker analysis as a typing system for Candida glabrata

Candida glabrata is one of the most important causes of nosocomial fungal infection. We investigated, using a multiplex PCR, three polymorphic microsatellite markers, RPM2, MTI, and ERG3, in order to obtain a rapid genotyping method for C. glabrata. One set of primers was designed for each locus, and one primer of each set was dye labeled to read PCR signals using an automatic sequencer. Eight reference strains including other Candida species and 138 independent C. glabrata clinical isolates were tested. The clinical isolates were collected from different anatomical sites of adult patients either hospitalized in different wards of two different hospitals or not hospitalized. Since C. glabrata is haploid, one single PCR product for each PCR set was obtained and assigned to an allele. The numbers of different alleles were 5, 7, and 15 for the RPM2, MTI, and ERG3 loci, respectively. The number of allelic associations was 21, leading to a discriminatory power of 0.84. The markers were stable after 25 subcultures, and the amplifications were specific for C. glabrata. A factorial correspondence analysis did not indicate any correlation between the 21 multilocus genotypes and the clinical data (source, sex, ward, anatomical sites). Microsatellite marker analysis is a rapid and reliable technique to investigate clinical issues concerning C. glabrata. However, its discriminatory power should be improved by testing other polymorphic microsatellite loci.     

Clamped homogeneous electric field gel electrophoresis typing of Torulopsis glabrata isolates causing nosocomial infections.

Thirty isolates of Torulopsis glabrata were examined by pulsed-field gel electrophoresis, which resolved 13 DNA pieces, allowing the identification of 12 types. Bands at 1,400, 1,200, 1,070, 1,025, 681, and 500 kbp were conserved. When applied to 18 isolates from an outbreak, 10 distinct types were identified by this technique. Seven patients had isolates which were identical.     

Random amplified polymorphic DNA typing versus pulsed-field gel electrophoresis for epidemiological typing of vancomycin-resistant enterococci.

Sixty vancomycin-resistant vanA mutant Enterococcus faecium (VRE) isolates, collected during a 40-month period from 48 patients hospitalized in a French Cancer Referral Center, were typed by using random amplified polymorphic DNA (RAPD), and the results were compared with those previously obtained by typing with SmaI pulsed-field gel electrophoresis (PFGE), which is currently recognized as the "gold standard." The discriminating power of RAPD typing, with seven primers and 11 combinations of primers, was tested on 18 strains, and only the most discriminating combination was further tested on the whole collection. We compared the epidemiological usefulness of RAPD typing of 60 clinical VRE isolates with that of SmaI PFGE typing. With primers AP4 and ERIC1R, RAPD generated 30 patterns versus the 36 patterns generated by SmaI PFGE. However, this did not hamper the epidemiologically correct clustering of 15 related strains and the detection of multiple colonization in nine patients. We conclude that this simple RAPD technique is well suited to the epidemiological typing of VRE and the monitoring of its nosocomial spread.     

Comparison of ribotyping and restriction enzyme analysis using pulsed-field gel electrophoresis for distinguishing Legionella pneumophila isolates obtained during a nosocomial outbreak.

Because of the ubiquity of Legionella isolates in aquatic habitats, epidemiologic evaluation of Legionella pneumophila strains is important in the investigation and subsequent control of nosocomial outbreaks of legionellosis. In this study, ribotyping and restriction enzyme analysis by pulsed-field gel electrophoresis (PFGE) were used to compare isolates of L. pneumophila obtained from patients and the environment during a nosocomial outbreak with unrelated control strains. Restriction enzyme analysis by PFGE resolved 14 different patterns among the L. pneumophila serogroup 1 and L. pneumophila serogroup 6 isolates involved in the study. Two of the patterns were observed in the three L. pneumophila serogroup 6 isolates from patients with confirmed nosocomial infections and environmental isolates from the potable water supply, which was, therefore, believed to be the source of the patients' infections. Three more patterns that were not present in isolates from patients with legionellosis were seen in isolates from the hospital environment, demonstrating the presence of multiple strains in the hospital environment. In the outbreak, one distinct pattern occurred among the L. pneumophila serogroup 1 isolates from patients with nosocomial infections, suggesting a common source; however, the source could not be determined. By comparison, ribotyping generated five patterns. However, some control strains of both L. pneumophila serogroups 1 and 6 possessed the same ribotypes as were present in the outbreak isolates. Both techniques were used successfully to subtype the isolates obtained during the investigation of the outbreak. Furthermore, restriction enzyme analysis by PFGE was useful for subdividing ribotypes and for distinguishing strains involved in the outbreak from epidemiologically unrelated strains.     

Evaluation of the discriminatory power of pulsed-field gel electrophoresis and PCR fingerprinting for epidemiologic typing of Candida species

Objective: To evaluate the discriminatory power of genotyping methods (PCR fingerprinting and pulsed-field gel electrophoresis) validated for Candida albicans in other Candida species.
Methods: Molecular typing methods are increasingly being applied for studies where the interpretation of data essentially relies on the typing results rather than epidemiologic data. In this situation, the discriminatory power (ability to identify differences among epidemiologically unrelated strains) of the typing method is important in allowing one to draw valid conclusions. By applying PCR fingerprinting, electrophoretic karyotyping, and restriction fragment endo-nuclease analysis using standard restriction enzymes and primers proven to be useful in previous studies, we evaluated whether the use of multiple genotyping methods is sufficient to delineate known unrelated strains among seven Candida species.
Results: All three methods identified individual genotypes for each of the seven Candida species studied. However, optimal strain delineation required the combined use of all three typing methods and was observed only within the small number of C. albicans and C. tropicalis isolates tested in this study.
Conclusion: Typing assays that are able to delineate a certain Candida species may not be used blindly for other species of that genus. Regarding the limited number of strains tested, further validation of the discriminative power of genotyping methods (including in C. tropicalis ) should be done.     

Comparison of pulsed-field gel electrophoresis and coagulase gene restriction profile analysis techniques in the molecular typing of Staphylococcus aureus

Pulsed-field gel electrophoresis (PFGE) and coagulase gene restriction profile (CRP) analysis techniques were used to analyze 71 Staphylococcus aureus isolates recovered from nine food-borne disease outbreaks. Twenty-two PFGE profiles and 11 CRPs were identified, with discrimination indices of 0.86 and 0.72, respectively. In addition, the variable regions of the coagulase genes of 39 isolates were sequenced and showed extensive identity, indicating that this is not an efficient alternative for the molecular typing of S. aureus.     

Comparison of ribotyping and pulsed-field gel electrophoresis for molecular typing of Acinetobacter isolates.

Seventy-three isolates of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex, including 26 isolates from 10 hospital outbreaks, were typed by ribotyping with EcoRI and ClaI and by pulsed-field gel electrophoresis (PFGE) of genomic DNA after digestion with ApaI. Ribotyping with EcoRI distinguished 31 ribopatterns. Digestion with ClaI generated another eight ribotypes. PFGE, in contrast, identified 49 distinct patterns with seven variants. Both methods detected all outbreak-related isolates. By ribotyping, nine epidemiologically unrelated strains could not be differentiated from outbreak strains, in contrast to only one isolate not identified by PFGE. Thus, PFGE was more discriminating than ribotyping. However, ribotyping is known to generate banding patterns specific to each DNA group in the A. calcoaceticus-A. baumannii complex that may be used for taxonomic identification of the strains. PFGE was shown to lack this property. Both methods are therefore useful for strain differentiation in epidemiological studies of Acinetobacter isolates.     

Comparison of pulsed-field gel electrophoresis and randomly amplified DNA polymorphism analysis for typing extended-spectrum-beta-lactamase-producing Klebsiella pneumoniae.

The incidence and transmission patterns of extended-spectrum-beta-lactamase (ESBL)-producing Klebsiella pneumoniae in patients admitted to the intensive care unit (ICU) of a university hospital were investigated over a 3-year period. K. pneumoniae isolates were characterized by antibiotic susceptibility, capsular serotyping, plasmid profiles, and pulsed-field gel electrophoresis (PFGE) of genome macrorestriction patterns with XbaI, and the results were compared with those obtained by typing with the randomly amplified polymorphic DNA (RAPD) patterns. The discriminatory power of RAPD typing was evaluated for three primers. The incidence of isolation of ESBL-producing K. pneumoniae was 2.5 cases per 1,000 admissions to the ICU versus 0.35 cases per 1,000 admissions to other units (relative risk, 7.03; 95% confidence interval, 3.89 to 12.69). Infection developed in 53% of evaluable patients. Thirty-six percent of the cases were possibly acquired in other institutions. Isolates from ICU patients were subdivided into six capsular serotypes and into four clonal groups based on antibiotype, plasmid content, and PFGE and RAPD patterns. Two clones were associated with clusters of cross-infection, involving 5 and 12 patients, respectively. Following implementation of contact isolation precautions, the incidence of nosocomial acquisition of ESBL-producing K. pneumoniae decreased from 0.55 to 0.26 cases per 1,000 admissions (P = 0.03). PFGE and RAPD analysis showed concordant results and comparable discrimination for differentiation between groups of epidemiologically related strains of ESBL-producing K. pneumoniae. More subclonal variants were determined among epidemic clones by PFGE analysis than by RAPD analysis. Both methods are useful for typing K. pneumoniae strains in epidemiological investigations, although RAPD analysis is more efficient.     

Comparison of multilocus sequence typing and pulsed-field gel electrophoresis as tools for typing Staphylococcus aureus isolates in a microepidemiological setting

Multilocus sequence typing (MLST) of Staphylococcus aureus is well suited to the study of global or long-term epidemiology, but its role in local epidemiology has not been defined. The present study has compared MLST with pulsed-field gel electrophoresis (PFGE) by using S. aureus isolates associated with carriage and disease in a busy regional renal unit. One hundred forty-four patients were prospectively recruited, of whom 103 were receiving hemodialysis and 41 were on continuous ambulatory peritoneal dialysis. Three nasal swab specimens were obtained 1 month apart on entering the study. A nasal swab was positive for S. aureus on at least one occasion in 50 patients (35%). Typing of the 104 carriage isolates demonstrated 21 PFGE types and 21 sequence types (STs). Thirty-one carriers had two or more positive nasal swabs; of these, the isolates in all swabs from a given carrier had identical PFGE types for 29 carriers; the isolates in all of the same 29 swabs had identical STs. The carriage strain in two patients changed both PFGE type and STs during the period of swabbing. Eight patients (6%) had an episode of S. aureus bacteremia during the 12-month study period, and two of these were nasal carriers. One of these invasive isolates had the same PFGE type and ST as the carriage isolate. There were no differences between Simpson's index of diversity for PFGE and Simpson's index of diversity for MLST for both invasive and carriage isolates, suggesting that the two methods have very similar discriminatory abilities. We conclude that PFGE and MLST performed equally in this study.     

Molecular probe for identification of medically important Candida species and Torulopsis glabrata.

A cloned DNA fragment from Candida albicans containing the gene for the protein actin was used to probe the molecular structure of the actin gene of several medically important yeasts (C. albicans, Candida stellatoidea, Candida tropicalis, Candida pseudotropicalis, Candida krusei, Candida parapsilosis, Candida guilliermondii, and Torulopsis glabrata). Whole-cell DNA from each species was digested with restriction endonucleases, electrophoresed on agarose gels, and transferred to nitrocellulose. Radioactively labeled C. albicans actin gene was hybridized to the DNA fragments on the nitrocellulose. The C. albicans probe produced a strong signal with all of the Candida DNAs tested, indicating considerable conservation of this gene. In addition, the actin genes of all of the species tested were found to have no internal EcoRI or SalI restriction sites. With the exception of C. guilliermondii, all of the species tested had a single internal HindIII recognition site. However, the location of flanking restriction sites was found to be species specific. For all of the enzymes tested, the locations of the flanking restriction sites in C. albicans and C. stellatoidea were identical; all of the other strains yielded fragments clearly distinct from one another. These differences provide a molecular tool for the differentiation of medically important Candida species.     

Comparison of an automated ribotyping system to restriction endonuclease analysis and pulsed-field gel electrophoresis for differentiating vancomycin-resistant Enterococcus faecium isolates

The RiboPrinter Microbial Characterization System was compared with pulsed-field gel electrophoresis (PFGE), restriction endonuclease analysis (REA), and epidemiological data for typing 45 vancomycin-resistant Enterococcus faecium (VRE) isolates. In 21 clinically related isolates, 90 to 100% were similar by PFGE and REA, but only 57% were similar by the RiboPrinter. In another eight clinically related isolates, three isolates similar by PFGE and REA were all unique by the RiboPrinter. In contrast, in 16 clinically unrelated isolates, the predominant RiboPrinter ribotype represented 50% of the strains, while the largest PFGE and REA clones represented less than 19% of the strains. These data suggest that the RiboPrinter is not reliable for VRE investigation.     

Comparison of molecular typing methods for Candida albicans.

Four molecular approaches to determining the types of Candida albicans strains were compared. The strains used were those whose repeated DNA (ribosomal and mitochondrial) EcoRI restriction fragment length polymorphisms (RFLP) were determined by Stevens et al. (D. A. Stevens, F. C. Odds, and S. Scherer, Rev. Infect. Dis. 12:258-266, 1990). Scherer and Stevens (S. Scherer and D. A. Stevens, Proc. Natl. Acad. Sci. USA 85:1452-1456, 1988) used the same strains to examine the Southern blots of genomic EcoRI digests probed with the repeated sequence 27A. The results of these investigators were compared with determinations of RFLPs generated from repeated DNA by the enzyme HinfI and examination of the karyotypes of strains under two sets of conditions, one for the smaller chromosomes and one for the larger ones. Analysis of RFLPs of repeated DNA is most convenient but shows the lowest degree of resolution. Use of the repeated sequence and use of karyotype have very high resolution, but the former method is more convenient than the latter. HinfI digestion is more sensitive than EcoRI digestion but equally convenient. By using all four methods, separate types were identified for 18 of the 20 strains examined.     

Multilocus sequence typing versus pulsed-field gel electrophoresis for characterization of extended-spectrum beta-lactamase-producing Escherichia coli isolates

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strains are emerging pathogens. Molecular typing of ESBL-producing E. coli is useful for surveillance purposes, to monitor outbreaks and track nosocomial spread. Although pulsed-field gel electrophoresis (PFGE) is the current "gold standard" for bacterial molecular typing, multilocus sequence typing (MLST) may offer advantages. Forty ESBL-producing E. coli isolates were selected at random from a cohort of intensive care unit patients who had active surveillance perirectal cultures done. PFGE identified 19 unique PFGE types (PT) among the 40 isolates; MLST identified 22 unique sequence types. MLST had greater discriminatory ability than PFGE for ESBL-producing E. coli. Simpson's indices of diversity for PFGE and MLST were 0.895 and 0.956, respectively. There were five clonal complexes (CCs) (isolates with differences of no more than two loci) that each contained multiple PT, but each PT was found in only one CC, indicating genetic consistency within a CC. MLST has clear utility in studies of ESBL-producing E. coli, based on a greater discriminatory ability and reproducibility than PFGE and the ability to a priori define genetically related bacterial strains.     

Epidemiological analysis of Candida albicans strains by multilocus enzyme electrophoresis.

Genotypic diversity in a collection of 98 isolates of Candida albicans was assessed by multilocus enzyme electrophoresis. Four of the 10 enzyme loci studied were polymorphic. The electrophoretic patterns observed were compatible with those expected for a diploid organism. The 98 isolates were assigned to 14 electrophoretic types, each of which was represented by from 1 to 21 isolates. Samples from various clinical sites of seven bone marrow transplant patients treated in the same unit within a 13-month period were obtained repeatedly. Three patients were found to be colonized with more than one strain. In one patient, flucytosine-resistant strains were isolated after systemic antifungal treatment was started. These isolates had electrophoretic types different from those of the strains that colonized the patient before treatment. There was no evidence that cross-infection between these patients occurred in the hospital. The population structure of C. albicans is discussed in regard to the multilocus genotype data.