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Genetic Diversity among Isolates of Aspergillus fumigatus in Patients with Cystic Fibrosis
Zbl. Bakt. 285, 450-455 (1997) © Gustav Fischer Verlag, Jena
Genetic Diversity among Isolates of Aspergillus fumigatus in Patients with Cystic Fibrosis * PETER-MICHAEL RATHI, FELIX RATJEN2, and RAINER ANSORG 1 I Institut fiir Medizinische Mikrobiologie der Universitiit-GH Essen, Germany Zentrum fiir Kinder- und Jugendmedizin der Universitii t-GH Essen, Germany
2
Received March 18, 1996 . Revision recei ved June 3, 1996 . Accepted July 9, 1996
Summary Strains of Aspergillus fumigatus (n = 24) were isolated from the sputa of six patients with cystic fibrosis during periods from 3 to 11 months. The genetic polymorph isms of the strains were studied using the random amplified polymorphic DNA (RAPD) assay with three single oligonucleotides and pairwise combined primers. The analysis of RAPD patterns resulted in 15 different RAPD types. In four patients, the colonizing type changed, whereas in two others the same types were detected over periods between 3 and 11 months. The genetic di versity as well as the shift of the colonizing strains found in some patients might be impor tant for the epidemiology of Aspergillus infections in patients with cystic fibrosis.
Introduction Cystic fibrosis (CF) is an autosomal recessive disorder occurring at a rate of appro ximately 112500 live births in the white population (3). Chronic lung infections in pa tients with CF are most frequently associated with Pseudomonas aeruginosa or Sta phylococcus au reus (4). Other common pathogens are Haemophilus in(luenzae and Burkholderia (Pseudomonas) cepacia (4) . The major fungal agent is Aspergillus fumi gatus. 60% to 80% of CF patients may be colonized with this fungus (4) . Except for individuals developing allergic bronchopulmonary aspergillosis (ABPA) (4, 11), little is known about the pathogenic significance of Aspergillus colonization (7). In additi on, no information is available on whether a colonizing Aspergillus strain may be re placed by another strain during the course of the illness. Recently, the RAPD assay was found to be a simple and rapid technique to diffe rentiate Aspergillus fumigatus strains in patients with invasive aspergillosis as well as with aspergilloma (1, 5, 6, 8), showing a high genetic diversity of the strains involved.
* The data were presented at the 47. KongreR der Deutschen Gesellschaft fiir Hygiene und Mikrobiologie (3.-7. 10. 1995, Wiirzburg, Germany) and the 9 th Annual North Ame rican Cystic Fibrosis Conference (12 .-15. 10. 1995, Dallas, USA).
Genetic Diversity of Aspergillus
451
In this PCR-based assay for DNA fingerprinting, single short oligonucleotides of ar bitrary sequence are used to amplify genomic DNA segments under low stringency conditions. By comparing the resulting patterns, it is possible to differentiate between strains (15). In the present study, 24 Aspergillus fumigatus isolates from six patients with cystic fibrosis were studied using the RAPD assay with three single oligonucleo tides and pairwise combined primers.
Materials and Methods Patients. Six patients with cystic fibrosis with repeatedly positive sputum cultures for As pergillus fumigatus were included in this study. None of the patients had clinical signs in dicating ABPA (10). The biometric data of the patients are listed in Table 1. The Shwach man score grades the clinical state of patients with cystic fibrosis. Points are allocated for general activity, physical findings, nutritional status and radiographic findings. A total of 100 points (25 points in each category) represents a perfect score (10). Aspergillus fumigatus isolates. Four to 11 sputum samples of each patient were cultured on standard media. In total, 24 Aspergillus fumigatus strains were isolated on Sabouraud dextrose agar (Unipath, UK) and were subsequently cultured on Sabouraud dextrose agar and Czapek-Dox agar (Difco, Detroit, USA). The isolates were classified on the basis of stan dard morphological features and the ability of growth at 48°C. Aspergillus fumigatus N CPF 2140 served as reference strain. Preparation of DNA and RAPD assay. Preparation of DNA and RAPD assay were per formed as previously described (8). Briefly, mycelial cells of the Aspergillus fumigatus strains, grown in Sabouraud dextrose liquid medium (Unipath, UK) for 24 h at 37°C were lysed by freezing and thawing and vortexing with glass beads. After treatment with extrac tion buffer (pH 8.0, 200 mM Tris-HCI, 0.5 M NaCl, 10 mM EDTA, 1 % (w/v) SDS), DNA was recovered by phenol-chloroform extraction and precipitation in ethanol. RNA was eli minated by RNase digestion in TE buffer (pH 7.9, 10 mM Tris-HCl, 1 mM EDTA). Con centrations of DNA were determined by using the DNA DipStick (Invitrogen, San Diego, USA). The oligonucleotides (primer 1:5'-GTATTGCCCT-3'; primer 2: 5'-GCTGGTGG-3'; primer 3: 5'-TCACCCTGCA-3') were the same as described before (8). DNA amplificati on was performed in a master mix (50mM KCl, 3mM MgCI 2 , 10mM Tris-HCl, pH 8.8), containing 200 !-1M (each) dATP, dCTP, dGTP and dTTP (BIOzym, Hameln, Germany),
Table 1. Biometric data of the patients with cystic fibrosis Patient
A B C D E F
Sex
female male male female female female
Age (years) 35 10 10 28 32 26
Colonized" with
P. aeruginosa
FVCb FEV!' (in % predicted)
75 50 85 60 65 68
92 50 83 50 44 60
S.aureus
+ + + + +
Shwachman score
+ +
48 31 33 23 48 24
" cultures from sputum samples: Pseudomonas aeruginosa and/or Staphylococcus aureus detected (+) or not detected (-). b forced vital capacity. C forced expiratory volume in one second.
452
P.-M. Rath, F. Ratjen, and R. Ansorg
1 ~g primer, 2.5 U of Taq DNA polymerase (Promega) and 5 ng of template DNA, using a Minicycler (MJ Research, Watertown, USA). In experiments with pairwise combined pri mers, 0.5 ~g of each primer was used. The cycles were as follows: One cycle at 94°C (5 min); 45 cycles at 94 °C (1 min), 35°C (2 min), and 72 °C (2 min); one cycle at 72 °C (10min). The amplified products were separated in a 0.8% agarose gel and stained with ethidium bromide. The gels were photographed under UV light. A 100 bp ladder (Pharma cia, Broma, Sweden) was run in parallel in each experiment. Each strain was investigated twice. Only major bands (intensity equal to or stronger than the 0.8 kb band of the mar ker) were included in the analysis. Patterns with differences in one or more major bands were designated as individual strains.
Results Aspergillus fumigatus strains were cultured from 24 out of 34 sputum samples of six patients with CF (Table 2). Four patients were investigated at intervals of two to four months. In patient E, the first four isolates were recovered within three weeks, whereas the fourth was isolated from a sputum sample obtained three months later. In patient B, the interval was three months between the first and the second investigation but two to three weeks between the third and fourth investigation. The 25 isolates stu died (including the reference strain) showed no differences in their macroscopic or microscopic morphology and all grew at 48°C. In the RAPD assay, each of the primers generated RAPD patterns with one to seven bands in the range of 0.28 to 1.8 kb. Primer 1 showed the highest discrimination with 12 different RAPD patterns within the 25 strains. No common band existing in all iso lates was seen. Primer 2 discriminated six different patterns. Common bands were found in the range of 1.4, 1.2, and 0.8 kb, whereas bands in the range of 0.65, 0.5, 0.32, and 0.28 kb varied between the isolates. Primer 3 showed the lowest discrimi nation with a single common band in the range of 0.6 kb and an additional band at 1.5 kb or at 0.5 kb. The combination of different patterns obtained by the three single primers resulted in 15 different RAPD types. In one patient, all five isolates showed
Table 2. Duration of study, number of sputum samples and Aspergillus fumigatus isolates, and results of RAPD analysis in six patients with cystic fibrosis Patient
A B C D E F
Duration of study (months)
No. of sputa
7 4 9 9 3 11
4 4 5 11 5 5
Aspergillus fumigatus
No. of isolates
Results of RAPD analysis
3 4 3 6 5 3
all different 2 different!2 identical all different 1 different!5 identical all identical all different
The results of RAPD analysis based on the patterns generated by three single primers. Iso lates were designated as different when one primer generated differences in one or more ma jor bands. Isolates showing identical patterns in the single primer assay were re-examined, using pairwise combined primers. Again, the resulting patterns were identical.
Genetic Diversity of Aspergillus
453
identical patterns in all primers (patient E). In patient D, one isolate was different in primers 1 and 3, whereas the five other isolates were identical in all primers. In the four other patients, each isolate exhibited different RAPD patterns. Only two isolates from different patients (patients A and C) showed identical RAPD patterns in all pri mers. The RAPD patterns of the isolates from patients A and C generated by primer 1 and primer 2 are shown in Fig. 1. Strains with identical RAPD patterns in the single primer RAPD assay were re-exa mined using pairwise combined primers. Again, no differences were found. The refe rence strain was different from the patient isolates. In one to five sputum samples from four patients, Aspergillus fumigatus was not iso lated (Table 2). Except in patient D, the strains from specimens recovered after nega tive samples had exhibited different RAPD patterns compared to the previous isolates. Discussion The 24 Aspergillus fumigatus strains recovered from sputum samples of six patients with cystic fibrosis (CF) showed no phenotypic differences. In contrast, analysis of the RAPD patterns generated by three single primers revealed 15 different types including the reference strains. Only two patients were transiently colonized with a strain sho wing identical patterns in the single primer assay as well as in the assay with pairwise combined primers. These data indicate that no common type of Aspergillus fumigatus colonized the patients. Furthermore, in four of the six patients, the colonizing types changed within the study period. These results are in contrast to data regarding the co lonization with Burkholderia cepacia (2) and Pseudomonas aeruginosa (9). In both Burkholderia cepacia and Pseudomonas aeruginosa genetic studies have shown a con-
A
B
Fig. 1. RAPD patterns of Aspergillus fumigatus isolates of two patients with cystic fibrosis, generated with primers 1 (A) and 2 (B). Lane 1: 100 bp ladder, lanes 2-4: isolates of patient A; lanes 5-7: isolates of patient C; lane 8: reference strain (NCPF 2140). The sizes of components of the 100 bp ladder are given on the left side. The comparison of the RAPD results revealed identical patterns of one isolate from patient A (lane 3) and one isolate from patient C (lane 6) in both primers.
454
P.-M. Rath, F. Ratjen, and R. Ansorg
stant colonization of individual patients with one or a few genotypes over long peri ods (up to eight years in Pseudomonas aeruginosa). In Burkholderia cepacia infection, only five genotypes were found in 23 isolates of 11 cystic fibrosis patients, indicating cross-infections or common environmental sources (2). Based on the data presented it is not possible to draw conclusions as to the duration of colonization with an Aspergillus fumigatus strain in an individual patient because the intervals between investigations were different, ranging from weeks to months. However, this duration seems to be highly variable between patients. In patients D and E, a constant colonization with one strain was found over a period of three and 11 months (only one of six isolates was different in patient D), whereas in patients A, B, C, and F, the type changed within one to three months. In this context it is interesting that the occurrence of culturaly negative sputum samples in three patients correlated with a shift of the colonizing strains. However, this was not found in patient D in whom the strain did not change. In this patient, it remained unclear whether the ne gative sputum samples were false-negative or the colonizing strain had been eliminat ed and recovered from environmental sources again. Furthermore, it could not be rul ed out that patients were colonized with more than one Aspergillus fumigatus strain at the same time. The interpretation of RAPD assay results is subject to some limitations. At first, the assay does not allow an identification of isolates on the species level. Theoretically, it is possible that different species exhibit identical RAPD patterns. Therefore, only pat terns of strains definitely identified as members of one species are comparable. Se condly, strains with identical pattern, generated with one or a few single oligonucleo tides might be different when re-tested with more primers. Recently, it has been de scribed that pairwise combined primers in the arbitrarily primed PCR (13) can result in more discrimination than the use of single oligonucleotides (14). This was also found in the RAPD assay with Aspergillus fumigatus strains isolated from immuno suppressed patients (8). However, when strains exhibiting identical RAPD patterns in the single primer assay were re-tested using pairwise combined primers, identical pat terns were seen again. These data indicated that the strains were truly identical. Acknowledgement. We thank D. Schmidt for excellent technical assistance.
References 1. Aufauvre-Brown, A., J. Cohen, and D. W. Holden: Use of randomly amplified polymor phic DNA markers to distinguish isolates of Aspergillus fumigatus. J. Clin. Microbiol. 30 (1992) 2991-2993 2. Bingen, E. H., M. Weber, J. Derelle, N. Brahimi, N. Y. Lambert-Zechovsky, M. Vidailhet, J. Navarro, and]. Elion: Arbitrarily primed polymerase chain reaction as a rapid method to differentiate crossed from independent Pseudomonas cepacia infections in cystic fi brosis patients. J. Clin. Microbio1.31 (1993) 2589-2593 3. Boucher, R. c.: Cystic fibrosis. In: Harrison's principles of internal medicine. 4th edition (K. J. Isselbacher, E. Braunwald, ]. D. Wilson, J. B. Martin, A. S. Fauci, D. L. Kasper, eds.), pp. 1194-1197. McGraw-Hill Inc. New York (1994) 4 . Gilligan, P. H.: Microbiology of airway disease in patients with cystic fibrosis . Clin. Microbiol. Rev. 4 (1991) 35-51 5. Loudon, K. w., J. P. Burnie, A. P. Coke, and R. C. Matthews: Application of polymerase chain reaction to fingerprinting Aspergillus fumigatus by random amplification of poly morphic DNA. J. Clin. Microbio1.31 (1993) 1117-1121
Genetic Diversity of Aspergillus
455
6. Loudon, K. w., A. P. Coke,]. P. Burnie, G. S. Lucas, and]. A. Liu Yin: Invasive asper gillosis: clusters and sources? J. Med. Vet. Mycol. 32 (1994) 217-224 7. Mroueh, S. and A. Spock: Allergic bronchopulmonary aspergillosis in patients with cy stic fibrosis. Chest 105 (1994) 32-36 8. Rath, P.-M., G. Marggraf, H. Dermoumi, and R. Ansorg: Use of phenotypic and geno typic fingerprinting methods in the strain identification of Aspergillus fumigatus. my coses 38 (1995) 429-434 9. Romling, U., B. Fiedler,]. Bo(5hammer, D. Grothues,]. Greipel, H. von der Hardt, and B. Tummler: Epidemiology of chronic Pseudomonas aeruginosa infections in cystic fi brosis. J. Infect. Dis. 170 (1994) 1616-1621 10. Shwachman, H. and L. L. Kulczycki: Long-term study of one hundred five patients with cystic fibrosis. Am. J. Dis. Child. 96 (1958) 6-15 11. Simmonds, E.].,]. M. Littlewood, and E. G. V. Evans: Cystic fibrosis and allergic bron chopulmonary aspergillosis. Arch. Dis. Child. 65 (1990) 507-511 12. Tang, C. M.,]. Cohen, A.]. Rees, and D. W. Holden: Molecular epidemiological study of invasive pulmonary aspergillosis in a renal transplantation unit. Eur. ]. Clin. Micro bioI. Infect. Dis. 13 (1994) 318-321 13. Welsh,]. and M. McClelland: Fingerprinting genomes using PCR with arbitrary primers. Nucl. Acid Res. 18 (1990) 7213-7218 14. Welsh,]. and M. McClelland: Genomic fingerprinting using arbitrarily primed PCR and a matrix of pairwise combinations of primers. Nucl. Acid Res. 19 (1991) 5275-5279 15. Williams,]. G. K., A. R. Kubelik, K.]. Livak,]. A. Rafalski, and S. V. Tingey: DNA po lymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl. Acid Res. 18 (1990) 6531-6535
Dr. med. Peter-Michael Rath, Institut fur Medizinische Mikrobiologie, Universitat-GH Essen, Hufelandstrage 55, D-45147 Essen, Germany, Tel.: 0201/723-3538, Fax: 0201/7233534
Genetic Diversity among Isolates of Aspergillus fumigatus in Patients with Cystic Fibrosis * PETER-MICHAEL RATHI, FELIX RATJEN2, and RAINER ANSORG 1 I Institut fiir Medizinische Mikrobiologie der Universitiit-GH Essen, Germany Zentrum fiir Kinder- und Jugendmedizin der Universitii t-GH Essen, Germany
2
Received March 18, 1996 . Revision recei ved June 3, 1996 . Accepted July 9, 1996
Summary Strains of Aspergillus fumigatus (n = 24) were isolated from the sputa of six patients with cystic fibrosis during periods from 3 to 11 months. The genetic polymorph isms of the strains were studied using the random amplified polymorphic DNA (RAPD) assay with three single oligonucleotides and pairwise combined primers. The analysis of RAPD patterns resulted in 15 different RAPD types. In four patients, the colonizing type changed, whereas in two others the same types were detected over periods between 3 and 11 months. The genetic di versity as well as the shift of the colonizing strains found in some patients might be impor tant for the epidemiology of Aspergillus infections in patients with cystic fibrosis.
Introduction Cystic fibrosis (CF) is an autosomal recessive disorder occurring at a rate of appro ximately 112500 live births in the white population (3). Chronic lung infections in pa tients with CF are most frequently associated with Pseudomonas aeruginosa or Sta phylococcus au reus (4). Other common pathogens are Haemophilus in(luenzae and Burkholderia (Pseudomonas) cepacia (4) . The major fungal agent is Aspergillus fumi gatus. 60% to 80% of CF patients may be colonized with this fungus (4) . Except for individuals developing allergic bronchopulmonary aspergillosis (ABPA) (4, 11), little is known about the pathogenic significance of Aspergillus colonization (7). In additi on, no information is available on whether a colonizing Aspergillus strain may be re placed by another strain during the course of the illness. Recently, the RAPD assay was found to be a simple and rapid technique to diffe rentiate Aspergillus fumigatus strains in patients with invasive aspergillosis as well as with aspergilloma (1, 5, 6, 8), showing a high genetic diversity of the strains involved.
* The data were presented at the 47. KongreR der Deutschen Gesellschaft fiir Hygiene und Mikrobiologie (3.-7. 10. 1995, Wiirzburg, Germany) and the 9 th Annual North Ame rican Cystic Fibrosis Conference (12 .-15. 10. 1995, Dallas, USA).
Genetic Diversity of Aspergillus
451
In this PCR-based assay for DNA fingerprinting, single short oligonucleotides of ar bitrary sequence are used to amplify genomic DNA segments under low stringency conditions. By comparing the resulting patterns, it is possible to differentiate between strains (15). In the present study, 24 Aspergillus fumigatus isolates from six patients with cystic fibrosis were studied using the RAPD assay with three single oligonucleo tides and pairwise combined primers.
Materials and Methods Patients. Six patients with cystic fibrosis with repeatedly positive sputum cultures for As pergillus fumigatus were included in this study. None of the patients had clinical signs in dicating ABPA (10). The biometric data of the patients are listed in Table 1. The Shwach man score grades the clinical state of patients with cystic fibrosis. Points are allocated for general activity, physical findings, nutritional status and radiographic findings. A total of 100 points (25 points in each category) represents a perfect score (10). Aspergillus fumigatus isolates. Four to 11 sputum samples of each patient were cultured on standard media. In total, 24 Aspergillus fumigatus strains were isolated on Sabouraud dextrose agar (Unipath, UK) and were subsequently cultured on Sabouraud dextrose agar and Czapek-Dox agar (Difco, Detroit, USA). The isolates were classified on the basis of stan dard morphological features and the ability of growth at 48°C. Aspergillus fumigatus N CPF 2140 served as reference strain. Preparation of DNA and RAPD assay. Preparation of DNA and RAPD assay were per formed as previously described (8). Briefly, mycelial cells of the Aspergillus fumigatus strains, grown in Sabouraud dextrose liquid medium (Unipath, UK) for 24 h at 37°C were lysed by freezing and thawing and vortexing with glass beads. After treatment with extrac tion buffer (pH 8.0, 200 mM Tris-HCI, 0.5 M NaCl, 10 mM EDTA, 1 % (w/v) SDS), DNA was recovered by phenol-chloroform extraction and precipitation in ethanol. RNA was eli minated by RNase digestion in TE buffer (pH 7.9, 10 mM Tris-HCl, 1 mM EDTA). Con centrations of DNA were determined by using the DNA DipStick (Invitrogen, San Diego, USA). The oligonucleotides (primer 1:5'-GTATTGCCCT-3'; primer 2: 5'-GCTGGTGG-3'; primer 3: 5'-TCACCCTGCA-3') were the same as described before (8). DNA amplificati on was performed in a master mix (50mM KCl, 3mM MgCI 2 , 10mM Tris-HCl, pH 8.8), containing 200 !-1M (each) dATP, dCTP, dGTP and dTTP (BIOzym, Hameln, Germany),
Table 1. Biometric data of the patients with cystic fibrosis Patient
A B C D E F
Sex
female male male female female female
Age (years) 35 10 10 28 32 26
Colonized" with
P. aeruginosa
FVCb FEV!' (in % predicted)
75 50 85 60 65 68
92 50 83 50 44 60
S.aureus
+ + + + +
Shwachman score
+ +
48 31 33 23 48 24
" cultures from sputum samples: Pseudomonas aeruginosa and/or Staphylococcus aureus detected (+) or not detected (-). b forced vital capacity. C forced expiratory volume in one second.
452
P.-M. Rath, F. Ratjen, and R. Ansorg
1 ~g primer, 2.5 U of Taq DNA polymerase (Promega) and 5 ng of template DNA, using a Minicycler (MJ Research, Watertown, USA). In experiments with pairwise combined pri mers, 0.5 ~g of each primer was used. The cycles were as follows: One cycle at 94°C (5 min); 45 cycles at 94 °C (1 min), 35°C (2 min), and 72 °C (2 min); one cycle at 72 °C (10min). The amplified products were separated in a 0.8% agarose gel and stained with ethidium bromide. The gels were photographed under UV light. A 100 bp ladder (Pharma cia, Broma, Sweden) was run in parallel in each experiment. Each strain was investigated twice. Only major bands (intensity equal to or stronger than the 0.8 kb band of the mar ker) were included in the analysis. Patterns with differences in one or more major bands were designated as individual strains.
Results Aspergillus fumigatus strains were cultured from 24 out of 34 sputum samples of six patients with CF (Table 2). Four patients were investigated at intervals of two to four months. In patient E, the first four isolates were recovered within three weeks, whereas the fourth was isolated from a sputum sample obtained three months later. In patient B, the interval was three months between the first and the second investigation but two to three weeks between the third and fourth investigation. The 25 isolates stu died (including the reference strain) showed no differences in their macroscopic or microscopic morphology and all grew at 48°C. In the RAPD assay, each of the primers generated RAPD patterns with one to seven bands in the range of 0.28 to 1.8 kb. Primer 1 showed the highest discrimination with 12 different RAPD patterns within the 25 strains. No common band existing in all iso lates was seen. Primer 2 discriminated six different patterns. Common bands were found in the range of 1.4, 1.2, and 0.8 kb, whereas bands in the range of 0.65, 0.5, 0.32, and 0.28 kb varied between the isolates. Primer 3 showed the lowest discrimi nation with a single common band in the range of 0.6 kb and an additional band at 1.5 kb or at 0.5 kb. The combination of different patterns obtained by the three single primers resulted in 15 different RAPD types. In one patient, all five isolates showed
Table 2. Duration of study, number of sputum samples and Aspergillus fumigatus isolates, and results of RAPD analysis in six patients with cystic fibrosis Patient
A B C D E F
Duration of study (months)
No. of sputa
7 4 9 9 3 11
4 4 5 11 5 5
Aspergillus fumigatus
No. of isolates
Results of RAPD analysis
3 4 3 6 5 3
all different 2 different!2 identical all different 1 different!5 identical all identical all different
The results of RAPD analysis based on the patterns generated by three single primers. Iso lates were designated as different when one primer generated differences in one or more ma jor bands. Isolates showing identical patterns in the single primer assay were re-examined, using pairwise combined primers. Again, the resulting patterns were identical.
Genetic Diversity of Aspergillus
453
identical patterns in all primers (patient E). In patient D, one isolate was different in primers 1 and 3, whereas the five other isolates were identical in all primers. In the four other patients, each isolate exhibited different RAPD patterns. Only two isolates from different patients (patients A and C) showed identical RAPD patterns in all pri mers. The RAPD patterns of the isolates from patients A and C generated by primer 1 and primer 2 are shown in Fig. 1. Strains with identical RAPD patterns in the single primer RAPD assay were re-exa mined using pairwise combined primers. Again, no differences were found. The refe rence strain was different from the patient isolates. In one to five sputum samples from four patients, Aspergillus fumigatus was not iso lated (Table 2). Except in patient D, the strains from specimens recovered after nega tive samples had exhibited different RAPD patterns compared to the previous isolates. Discussion The 24 Aspergillus fumigatus strains recovered from sputum samples of six patients with cystic fibrosis (CF) showed no phenotypic differences. In contrast, analysis of the RAPD patterns generated by three single primers revealed 15 different types including the reference strains. Only two patients were transiently colonized with a strain sho wing identical patterns in the single primer assay as well as in the assay with pairwise combined primers. These data indicate that no common type of Aspergillus fumigatus colonized the patients. Furthermore, in four of the six patients, the colonizing types changed within the study period. These results are in contrast to data regarding the co lonization with Burkholderia cepacia (2) and Pseudomonas aeruginosa (9). In both Burkholderia cepacia and Pseudomonas aeruginosa genetic studies have shown a con-
A
B
Fig. 1. RAPD patterns of Aspergillus fumigatus isolates of two patients with cystic fibrosis, generated with primers 1 (A) and 2 (B). Lane 1: 100 bp ladder, lanes 2-4: isolates of patient A; lanes 5-7: isolates of patient C; lane 8: reference strain (NCPF 2140). The sizes of components of the 100 bp ladder are given on the left side. The comparison of the RAPD results revealed identical patterns of one isolate from patient A (lane 3) and one isolate from patient C (lane 6) in both primers.
454
P.-M. Rath, F. Ratjen, and R. Ansorg
stant colonization of individual patients with one or a few genotypes over long peri ods (up to eight years in Pseudomonas aeruginosa). In Burkholderia cepacia infection, only five genotypes were found in 23 isolates of 11 cystic fibrosis patients, indicating cross-infections or common environmental sources (2). Based on the data presented it is not possible to draw conclusions as to the duration of colonization with an Aspergillus fumigatus strain in an individual patient because the intervals between investigations were different, ranging from weeks to months. However, this duration seems to be highly variable between patients. In patients D and E, a constant colonization with one strain was found over a period of three and 11 months (only one of six isolates was different in patient D), whereas in patients A, B, C, and F, the type changed within one to three months. In this context it is interesting that the occurrence of culturaly negative sputum samples in three patients correlated with a shift of the colonizing strains. However, this was not found in patient D in whom the strain did not change. In this patient, it remained unclear whether the ne gative sputum samples were false-negative or the colonizing strain had been eliminat ed and recovered from environmental sources again. Furthermore, it could not be rul ed out that patients were colonized with more than one Aspergillus fumigatus strain at the same time. The interpretation of RAPD assay results is subject to some limitations. At first, the assay does not allow an identification of isolates on the species level. Theoretically, it is possible that different species exhibit identical RAPD patterns. Therefore, only pat terns of strains definitely identified as members of one species are comparable. Se condly, strains with identical pattern, generated with one or a few single oligonucleo tides might be different when re-tested with more primers. Recently, it has been de scribed that pairwise combined primers in the arbitrarily primed PCR (13) can result in more discrimination than the use of single oligonucleotides (14). This was also found in the RAPD assay with Aspergillus fumigatus strains isolated from immuno suppressed patients (8). However, when strains exhibiting identical RAPD patterns in the single primer assay were re-tested using pairwise combined primers, identical pat terns were seen again. These data indicated that the strains were truly identical. Acknowledgement. We thank D. Schmidt for excellent technical assistance.
References 1. Aufauvre-Brown, A., J. Cohen, and D. W. Holden: Use of randomly amplified polymor phic DNA markers to distinguish isolates of Aspergillus fumigatus. J. Clin. Microbiol. 30 (1992) 2991-2993 2. Bingen, E. H., M. Weber, J. Derelle, N. Brahimi, N. Y. Lambert-Zechovsky, M. Vidailhet, J. Navarro, and]. Elion: Arbitrarily primed polymerase chain reaction as a rapid method to differentiate crossed from independent Pseudomonas cepacia infections in cystic fi brosis patients. J. Clin. Microbio1.31 (1993) 2589-2593 3. Boucher, R. c.: Cystic fibrosis. In: Harrison's principles of internal medicine. 4th edition (K. J. Isselbacher, E. Braunwald, ]. D. Wilson, J. B. Martin, A. S. Fauci, D. L. Kasper, eds.), pp. 1194-1197. McGraw-Hill Inc. New York (1994) 4 . Gilligan, P. H.: Microbiology of airway disease in patients with cystic fibrosis . Clin. Microbiol. Rev. 4 (1991) 35-51 5. Loudon, K. w., J. P. Burnie, A. P. Coke, and R. C. Matthews: Application of polymerase chain reaction to fingerprinting Aspergillus fumigatus by random amplification of poly morphic DNA. J. Clin. Microbio1.31 (1993) 1117-1121
Genetic Diversity of Aspergillus
455
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Dr. med. Peter-Michael Rath, Institut fur Medizinische Mikrobiologie, Universitat-GH Essen, Hufelandstrage 55, D-45147 Essen, Germany, Tel.: 0201/723-3538, Fax: 0201/7233534