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Epidemiology of chronic Pseudomonas aeruginosa infections in cystic fibrosis
IJMM IJ
Int. J. Med. Microbiol. 291, 387-393 (2001) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ijmm
Epidemiology of chronic Pseudomonas aeruginosa infections in cystic fibrosis Burçin S¸ener1, Özgen Köseog˘lu1, Ug˘ur Özçelik2, Tanıl Kocagöz1, Ayfer Günalp1 1 2
Hacettepe University, School of Medicine, Department of Microbiology and Clinical Microbiology, Ankara, Turkey Hacettepe University, School of Medicine, ˙I hsan Dog˘ramacı Children’s Hospital, Department of Chest Diseases, Ankara, Turkey
Received June 1, 2001 · Revision received July 26, 2001 · Accepted July 26, 01
Abstract Chronic lung infection with Pseudomonas aeruginosa is primarily responsible for pulmonary deterioration of cystic fibrosis patients. The purpose of this study was to type the P. aeruginosa isolates collected sequentially from cystic fibrosis patients, chronically colonized with P. aeruginosa, by random amplified polymorphic DNA fingerprinting-PCR (RAPD-PCR). Sequential P. aeruginosa isolates (n: 130) that had been collected from 20 CF patients over at least 9 years were investigated. The isolates were analyzed by RAPD-PCR using two arbitrary primers. Antimicrobial susceptibility testing of all isolates was performed by the disc diffusion method. RAPD-PCR typing demonstrated that strains dissimilar in colony morphotype and of different antibiotic susceptibility patterns could be of the same genotype. Some CF patients were colonized with a rather constant P. aeruginosa flora, with strains of different phenotypes but of one genotype. However, some patients may be colonized with more than one genotype. The results also demonstrated that there might be a risk of cross-colonization between CF patients followed-up at the same center. Key words: Cystic fibrosis – Pseudomonas aeruginosa – Random amplified polymorphic DNA fingerprinting (RAPD-PCR)
Introduction Chronic lung infection with Pseudomonas aeruginosa is primarily responsible for the pulmonary deterioration and reduced life expectancy in patients with cystic fibrosis (CF) (Gilligan, 1991; Gowan and Deretic, 1996). Once P. aeruginosa has taken residence in the CF lungs, it is rarely possible to eradicate it by antimicrobial chemotherapy (Gowan and Deretic, 1996). Analysis of such therapeutic failure requires distinction between persistence of the same strain with the same
susceptibility pattern, emergence of resistance in the same strain, or reinfection with a new strain. Due to the difficulties in typing isolates of P. aeruginosa from CF patients by conventional phenotypic typing techniques, the epidemiology of P. aeruginosa infections in CF patients is still not well defined. Genetic typing methods have shown to be more discriminatory than phenotypic methods for typing P. aeruginosa CF isolates (Grundmann et al., 1995; Mahenthiralingam et al., 1996; The International Pseudomonas aeruginosa Typing Study Group, 1994). Molecular
Corresponding author: B. Óener, Hacettepe University, School of Medicine, Department of Microbiology and Clinical Microbiologya, 06 100-Sıhhiye Ankara, Turkey, Phone: +90 312 3 051 560, Fax: +90 312 3 115 250, E-mail: [email protected] 1438-4221/01/291/5-387 $ 15.00/0
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typing of P. aeruginosa CF isolates revealed the persistence of single clones in some CF patients (Bingen et al., 1992; Fegan et al., 1991; Mahenthiralingam et al., 1996; Römling et al., 1994) and also co-colonization with one or more strains (Fegan et al., 1991; Mahenthiralingam et al., 1996). With the recent developments in genotyping techniques it became possible to reexamine the strains and to provide a definitive conclusion. PCR based fingerprinting (random amplified polymorphic DNA fingerprinting-PCR, RAPD-PCR) was applied in this study since it has already proven its value for the genotypic characterization of many medically important bacteria including P. aeruginosa (Belkum, 1994; Kersulyte et al., 1995). This study was conducted to type the P. aeruginosa isolates collected sequentially from 20 CF patients, to determine whether a single strain or multiple strains were responsible for chronic colonization and also to detect if there was any relation between the changes in the antimicrobial susceptibility pattern and the genotype of the isolates, and also to determine whether any cross-colonization/infection exists between the patients. We also tried to provide a conclusive answer for the risk of cross-infection between these CF patients.
Materials and methods Patients and isolates The epidemiology of chronic colonization of the airways with P. aeruginosa was studied for a 10-year period (1991– 2000), in 20 patients with CF attending Hacettepe University ˙I hsan Dog˘ramacı Children’s Hospital Paediatric Chest Diseases Unit. For each patient, the diagnosis of CF had been suspected by typical clinical findings of pulmonary and gastrointestinal disease and was subsequently confirmed by pilocarpine iontophoresis sweat tests. A total of 130 strains out of a total of 248 strains were included into the study. These 130 strains belonged to 20 patients who were selected out of 53 CF patients on the basis of colonization/infection for more than at least 6 months with P. aeruginosa. The remaining 33 patients had only 1 P. aeruginosa strain isolated during the study period. If the number of P. aeruginosa strains for a patient was more than 10, then 10 of these isolates were selected for genotype analysis according to the following criteria: 1) Simultaneous isolates of different colony morphotypes or antibiotic resistance patterns in one sputum sample, were taken as different strains. 2) Care was taken to select one strain as the representative of a certain morphotype or antibiotic resistance pattern during the follow-up for each patient. The follow-up periods of the selected cases, their age, sex characteristics, the colony morphotype changes, the changes in antibiotic resistance patterns, and the RAPD-PCR genotypes are shown in Table 1. P. aeruginosa strains were isolated from sputa or deepthroat swab specimens. Sputum cultures were performed at
regular intervals (monthly) for each patient (if the patient applied to the hospital for control) and also during infective exacerbations. The diagnosis of chronic P. aeruginosa infection was based upon the detection of P. aeruginosa in sputum for a period of at least 6 consecutive months. Isolates were identified as P. aeruginosa on the basis of typical morphology, positive oxidase reaction, ability to produce pigments, growth at 42 °C, and oxidative utilization of glucose (Gilligan, 1995). Colony morphologies were assessed as by Wahba and Darrell (1965), as mucoid, classic and rough. CF patients were called to the Chest Diseases Unit (an outpatient clinic) on the last Tuesday of each month for their routine control visits. These patients waited for 1–3 hours in the same waiting-room without wearing face-masks during these control visits. The approximate area of the waiting room is 15 m2 and the approximate distance of the patients varies between 1 to 5 m. The patients with or without P. aeruginosa infection/colonization were called for routine control visits without regarding P. aeruginosa infection. Environmental sampling was carried out from the single sink drain and the tap water in that room. The patients do not routinely wash their hands in this sink. All dry sites were sampled with sterile swabs moistened with sterile saline. The carriage of P. aeruginosa on staff hands was tested by taking samples from the staff examining these patients on that day. There was no instrument, or other area shared commonly by these patients. They used their own nebulizers for inhalatory treatment and the mouth applicator of the pulmonary function test equipment was disposable. There was no washbasin in the physiotherapy or pulmonary function test rooms. The staff working in those rooms washed their hands at the washbasin found in a separate room which was routinely tested by the Hospital Infection Control Committee. Antimicrobial susceptibility testing of all isolates was done by Kirby-Bauer disc diffusion method according to the guidelines of National Committee for Clinical Laboratory Standards (NCCLS) (1997). The tested antimicrobial agents were as follows: Aztreonam, piperacillin, ceftazidime, cefepime, sulbactam cefoperazone, meropenem, imipenem, ciprofloxacin, amikacin, tobramycin. The strains were stored at –70 °C until processed for RAPD-PCR analysis. Isolation of P. aeruginosa genomic DNA A single colony of P. aeruginosa was inoculated into 2 ml of trypticase soy broth and grown overnight at 37 °C. The bacteria were pelleted in a 1.5-ml microcentrifuge tube by spinning at 12 000 g for 3 minutes. The cells were then washed three times with 750 µl TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), boiled for 20 minutes in 500 µl TE, centrifuged, and the supernatant containing the DNA was stored at –20 °C until RAPD-PCR was applied. RAPD- PCR analysis Fourteen different primers (ECGA2: 5-TGCGCTGCCAGATGTCCGAG-3, SJMT3: 5-CCTGTGGGGTCCGGCCTTTC, ECGAr1: 5-GATAGAACCGAAGTTACCCT-3, T3: 5-CAAGGACGCACATGACAGGA, SJMT1: 5-ACCACC-
Epidemiology of Pseudomonas aeruginosa isolates
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Table 1. Summary of phenotypic and genotypic typing results of P. aeruginosa isolates derived from all CF patients studied. Patient
Sex
Age at the time of study (yr)
Time (month)
Total number of isolates tested
Colony morphology
Genotype
1
M
9
16
2
C
1
2
S
2
M
11
12
2
R
1
2
F
3
F
17
16
2
R
1
2
A
4
F
7
26
2
C
1
2
S
5
M
3
6
2
C
2
2
S
6
M
6
24
3
R
1
3
S, A
7
M
11
22
4
C
3
4
S
8
F
9
34
4
C
1
3 1
S, B
4
Number of isolates with genotype
Antibiotic resistance pattern
B
9
M
3*
28
5
C, M
1
5
S
10
M
7
25
5
R, C
5
6
4 1
S A
11
F
6
34
7
R, M
7, 7 8 9
4, 1 1 1
A F F
12
M
17*
57
10
C, M, R
10
10
S, A, G,H
13
M
21
40
10
C, M
11
8 2
S, A, C
14
M
20*
14
10
M
12
13
9 1
C, G, E E
15
M
18
72
10
M, R
14 15
8 2
S S
16
F
13
26
10
R, M
16
10
A, H
17
M
13
24
10
R, M
17
10
S
18
F
14
66
10
R, C
18
16
8 2
S, B S
10
C
19
M
15
36
11
M, R
19 20 21
9 1 1
S, F S H
20
F
10
24
11
C, M, R
22 23 24
9 1 1
S S S
Colony morphology; M: Mucoid, C: Classic, R: Rough. *; Exitus.
Antibiotic resistance patterns; S: Susceptible to all antibiotics tested, A: Resistant to ceftazidime, aztreonam, B: Resistant to amikacin and/or tobramycin, C: Resistant to ceftazidime, amikacin, D: Resistant to ceftazidime, amikacin, ciprofloxacin, E: Resistant to ceftazidime, amikacin, ciprofloxacin, imipenem, F: Resistant to tobramycin, imipenem, G: Resistant to ceftazidime, aztreonam, imipenem, H: Resistant to ceftazidime, aztreonam, imipenem and amikacin.
GAGCGGTTCGCCTGA-3, M1: 5-GAAGCTTATGGTACAGGTTGG, TT15: 5-TCGCGAATCAGCTCGCCG-3, SJMTr2: 5-GATCTGCGGGTCGTCCCAGGT-3, MT2: 5-CTCGTCCAGCGCCGCTTCGG-3, MTBGyrAend: 5TCACCCCGACTCCTAACACT-3, INHA1: 5-CCACAACTAGAATGCAGTGAAAAAA, M2: 5-ATTACCATCCTTGTTGTAAG, L2: 5-CTGGCTTCTTCCAGCTTCA-3, TT16: 5-GCTTTGCAAGCTCCTCACC-3) of 18–26 nucleotides, used for amplification of various genes of several other microorganisms, were initially screened for reproduc-
ibility and the ability to produce discriminatory polymorphisms by using DNA of P. aeruginosa ATCC 27 853. Four primers (SJMT3, M1, T3 and INHA1) were found to result in reproducible and discriminatory amplification of DNA fingerprints; primer SJMT3 was selected for primary typing of the CF isolates described in this study, and primer M1 was used for confirmation. SJMT3 and M1 were selected upon their higher GC content. Each strain was typed twice under the same conditions to test reproducibility and reliability of the results.
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RAPD-PCR mixtures of 25 µl, containing 1 reaction buffer, 2.5 mM MgCl2, 0.2 mM (each) deoxynucleosidetriphosphates (dNTP), 0.65 U Replitherm polymerase (Epicentre Technologies), 30 pM primer and 2 µl sample DNA were prepared. The DNA was denatured for 3 min at 94 °C. At the beginning, 3 cycles were performed with an automated thermal cycler (MJR Research) at low annealing temperature. Each cycle consisted of denaturation at 94 °C for 1 min, annealing of primers at 30 °C for 1 min and primer extension at 72 °C for 1 min. Then 45 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min, followed by a final extension step at 72 °C for 3 min were performed. RAPD-PCR products (10 ml of each reaction mixture) were then separated by electrophoresis in 1.5 % agarose gels with 1 TAE running buffer. Gels were stained with ethidium bromide and visualized on a UV transilluminator and photographed. The bands were further examined by Ultra Violet Products (UVP) White/UV transilluminator and GrabIT annotating grabber program. Gel images were then analyzed by GelWorks ID Intermediate Version 2.51 (Nonlinear Dynamics Ltd) according to the manufacturer’s instructions.
First, sample and molecular weight marker lanes were located manually. After automatic band detection, a visual inspection was carried out in order to determine the unrecognized bands. For each gel, φx174 was used as molecular weight standard to calculate the absolute lengths of the bands in individual sample lanes. Those calculations in turn, yielded numeric band patterns that are suitable for the evaluation of each strain. Each unique band pattern was denoted with letters from “1 to 24”. Very closely related genotypes in which only sub-band differences were found were coded by the same number with an additive (7 versus 7).
Results The 20 patients taken into the study yielded a total of 248 P. aeruginosa strains and among these 130 strains were genotyped according to the criteria mentioned above. The results of environmental and clinical staff screening performed by us and by the routine Hospital Infection Control Committee revealed no P. aeruginosa isolation. Selection of primers for RAPD-PCR analysis of P. aeruginosa
Fig. 1. P. aeruginosa ATCC 27853 strain-specific DNA polymorphisms generated by 14 different AP-PCR primers. Molecular size markers were run in lane M and their sizes (in base pairs) are as follows: 1353, 1078, 872, 603, 310, 281. The primers tested were as follows, in order of the lanes above (1– 14): ECGA2, SJMT3, ECGAr1, T3, SJMT1, M1, TT15, SJMTr2, MT2, MTBGyrAend, INHA1, M2, L2, TT16.
Fig. 2. The common RAPD genotype 1, shared by the patients (6, 2, 3, 4, 9, 1 and 8), generated by the primer M1. The first lane denoted by “M” is the molecular weight marker φx174.
Among the 14 primers tested, 4 were found to result in reproducible and discriminatory amplification polymorphisms suitable for strain differentiation of P. aeruginosa. The P. aeruginosa strain-specific polymorphisms generated by different RAPD-PCR primers are shown in Figure 1. The suitable primers were all of high GC content. SJMT3 and M1 were selected since they produced more bands than the other primers tested. Each primer yielded RAPD-PCR patterns which enabled the differentiation of unique strains of P. aeruginosa. The RAPD-PCR patterns revealing bands with different numbers, sizes and intensities were considered to represent different isolates. RAPD-PCR amplification patterns obtained from the 20 patients revealed 24 unique RAPD types (Table 1). Seven patients (patients 1, 2, 3, 4, 6, 8, 9) shared the same genotype, denoted by 1, the strains being isolated between 1992–2000, and possibly originated from patient 9 (strain first isolated in May 1992) (Figure 2). Of these seven patients, six patients (patients 1, 3, 4, 5, 6 and 9) harbored this genotype consistently within their lungs during repeated sampling over a 12months period. However, patient 8 was found to harbor a second genotype in only one sample besides the common shared one. Besides these 7 patients, patients 12 and 13, and patients 16 and 18 shared common genotypes, genotypes 10 and 16, respectively. Fifteen patients had P. aeruginosa isolates with one to three different genotypes. Among the 20 patients, 13 exhibited
Epidemiology of Pseudomonas aeruginosa isolates
a single persistent colonizing genotype. A “persistent colonizing genotype” was defined as the same genotype isolated from all specimens (at least 5 specimens) of a patient collected over the study period. “Transient colonizing genotypes” were defined as those isolated only from one or two of the specimens collected during the study period, suggesting reinfection, or colonization. Nine patients (patients 8, 10, 11, 13, 14, 15, 18, 19, 20) showed transient colonization with genotypes other than the persistent genotype. For example for the patient 11, the RAPD genotype of the strains collected between January 1997 and October 2000 are given in Figure 3, lanes 4 and 5 reveal the genotypes (7 and 7) of strains simultaneously isolated in the same sputum sample, with different colony morphotypes. Lanes 6 and 7 denote strains isolated in June 1999 (genotype 8) and October 2000 (genotype 9), respectively. Lane 8 belongs to genotype 7, which was the initial persistent genotype for that patient. The results revealed that the genotype 7 was the persistent genotype while the others (7, 8 and 9) represent transient colonization. Nine patients harbored more than one genotype during their whole follow-up and sometimes simultaneously i. e. two different patterns could be found in one sputum sample, e. g. patients 13, 15 and 18. The genotyping of sequential isolates from the nine patients, 12 through 20, revealed that each of these patients was chronically colonized/infected with strains of a single predominating RAPD type which remained stable for isolates collected over several years. For example, the first and the last isolates of patient 12 (RAPD type 10) were recovered 4.7 years apart, isolates from patient 15 (RAPD type 14) 6 years apart, isolates from patient 17 (RAPD type 17) 3 years apart (Figure 4). Six of these 9 patients were co-colonized at one time or another with two or more strains of different RAPD type, e. g. patient 19 was found to harbor isolates with RAPD types 19 and 20 at one visit. Many isolates identified as identical by RAPD genotype displayed a variable colony morphotype. For example isolates from patient 12 with RAPD type 10 exhibited classic, mucoid and rough colony forms at different isolation dates. Genotypically identical isolates also exhibited variability in antibiotic resistance pattern. For example, the isolates in RAPD type 10 for patient 12, RAPD type 11 for patient 13, RAPD type 12 for patient 14, RAPD type 18 for patient 18, RAPD type 19 for patient 19 revealed different antibiotic resistance profiles.
Discussion The poor prognosis of CF is especially associated with an early onset of P. aeruginosa lung infection. Typing
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Fig. 3. RAPD-PCR profile of the patient 11, generated by the primer M1. The first lane denoted by “M” is the molecular weight marker φx174. The strains were collected between January 1997 and October 2000. Lanes 4 and 5 reveal the genotypes (7 and 7) of strains simultaneously isolated in the same sputum sample, with different colony morphotypes. Lanes 6 and 7 denote strains isolated in June 1999 and October 2000, respectively.
Fig. 4. RAPD-PCR profile of patient 17, generated by the primer M1.The first lane denoted by “M” is the molecular weight marker φx174. The 10 sequential isolates collected between November 1996 and December 1999 show the same band pattern.
of P. aeruginosa isolates of CF origin by conventional phenotypic methods has been found to be difficult due to either the mucoid nature or the rough outer membrane of the isolates. Detailed analysis of P. aeruginosa populations in chronically infected CF patients is necessary, in particular to examine the complexity and stability of bacterial populations and to understand better how they adapt and evolve. More recently, genetic methods have been applied to study the epidemiology of P. aeruginosa in CF patients (Grundmann et al., 1995; The International Pseudomonas aeruginosa Typing Study Group, 1994). Of the many DNA-based typing methods, RAPD-PCR seemed to be an efficient and sensitive means of high-throughput typing of such strains (Belkum, 1994; Hoogkamp-Korstanje et al., 1995; Kersulyte et al., 1995). This longitudinal study revealed that most CF patients were harboring the same P. aeruginosa genotype
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from the onset of colonization throughout the whole follow-up period. Turnover of the predominant genotype was not seen in any of the cases. This result was also obtained in some other previous studies (Kersulyte et al., 1995; Römling et al., 1994). However, occasional transient, or consistent genotypes were recovered in some of the patients (patients 8, 10, 11, 13, 14, 15, 18, 19, 20), usually simultaneously with the recovery of the predominant genotype. Some researchers contributed the absence of any predominant genotype to the limited time course of the study (Fegan et al., 1991). This was not the case in this study, since most patients were followed-up for more than 24 months. In general, despite alterations in the colony morphotype and antibiotic resistance profile, the RAPD fingerprints of sequential isolates remained stable, suggesting that these colonizing isolates enhanced their capacity to survive in the lower respiratory tract of CF patients. The question of cross-colonization in CF patients has not been conclusively resolved (HoogkampKorstanje et al., 1995). The results of this study suggest that there is risk of cross-colonization between CF patients followed-up at the same CF center. It was observed that RAPD-type 1 was the most commonly shared P. aeruginosa genotype in this study population, being recovered in 7 patients. It is noteworthy that P. aeruginosa RAPD type 1 was first isolated in May 1992 in patient 9 and was still present in the isolates of some of the patients in 2000. The risk of crosscolonization in our CF center may be due to the fact that CF patients are called for routine follow-up to the center on every last Tuesday of the month, and are educated for physiotherapy at the same place. Since these patients used their own nebulizers for treatment and no P. aeruginosa was isolated from the hands of staff members, this route of transmission does not seem to be a major factor for cross-colonization in these cases. None of these patients were known to socialize together outside the hospital, and none of them were siblings. It had been shown by Doring et al. (1991) that aerosols containing P. aeruginosa are known to be generated during hand washing from colonized or infected patients. During this study, although no P. aeruginosa has been isolated from washbasins and tap water, the U-shaped tube of these sinks was not sampled. Therefore, since there might be a P. aeruginosa source in these tubes, according to our results it can be concluded that this route plus being together in a small, crowded room with cough and sneeze droplets seemed to be the only possible ways, although further studies to ascertain the exact mode of transmission of P. aeruginosa between CF cases seem to be necessary. It is a matter of debate why other CF patients in our center are not colonized with RAPD type 1. An explanation for this may be that the other patients’ initial coloniz-
ing genotypes may outcompete this common genotype within the lung environment. Like other investigators, we have found that molecular characterization is the most helpful tool for typing CF P. aeruginosa strains. We used RAPD-PCR because it has high discriminatory power and already has proven its value in epidemiological studies of medically important microorganisms including P. aeruginosa (Belkum, 1994). Data presented here show that some of the CF patients are colonized with a rather constant P. aeruginosa flora, with strains of different phenotypes but of one genotype over a long period of time. However, we also found patients colonized with more than one genotype simultaneously, suggesting that the composition of the flora is not constant as a rule, but it may fluctuate considerably. These results also suggest the presence of risk of cross-colonization among our CF patients, and as a precaution these patients are now being called for control visits on different days of the month and are told to wear face masks in the waiting room. Acknowledgement. We thank Dr. Doruk Engin for his technical help in analysis of gel images. This study was partly presented at the 22nd European CSConference, June 13–19 1998, Berlin.
References Belkum, A.: DNA fingerprinting of medically important microorganisms by use of PCR. Clin. Microb. Rev. 7, 174–184 (1994). Bingen, E., Denamur, E., Picard, B., Goullet, P., LambertZechovsky, N., Foucaud, P., Navarro, J., Elion, J.: Molecular epidemiological analysis of Pseudomonas aeruginosa strains causing failure of antibiotic therapy in cystic fibrosis patients. Eur. J. Clin. Microbiol. Infect. Dis. 11, 432– 437 (1992). Doring G, Ulrich, M., Muller, W., Bitzer, J., Schmidt-Koenig, L., Munst, L., Grupp, H., Wolz, C., Stern, M., Botzenhart, K.: Generation of P. aeruginosa aerosols during hand washing from contaminated sink drains, transmission to hands of hospital personnel, and its prevention by use of a new heating device. Zentralbl. Hyg. 191, 494–505 (1991). Fegan, M., Francis, P., Hayward, A. C., Fuerst, J. A.: Heterogeneity, persistence, and distribution of Pseudomonas aeruginosa genotypes in cystic fibrosis. J. Clin. Microbiol. 29, 2151–2157 (1991). Gilligan, P. H.: Microbiology of airway disease in patients with cystic fibrosis. Clin. Microbiol. Rev. 4, 35–51 (1991). Gilligan, P. H. : Pseudomonas and Burkholderia. In: Manual of clinical microbiology (P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, R. H. Yolken, eds.), pp. 509–519. American Society for Microbiology, Washington, D. C. 1995.
Epidemiology of Pseudomonas aeruginosa isolates Gowan, J. R. W., Deretic, V.: Microbial pathogenesis in cystic fibrosis: Mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol. Rev. 60, 539–574 (1996). Grundmann, H., Schneider, C., Hartung, D., Daschner, F. D., Pitt, D. L.: Discriminatory power of three DNA-based typing techniques for Pseudomonas aeruginosa. J. Clin. Microbiol. 33, 528–534 (1995). Hoogkamp-Korstanje, J. A. A., Meis, J. F. G. M., Kissing, J., van der Laag, J., Melchers, J. W. G.: Risk of crosscolonization and infection by Pseudomonas aeruginosa in a holiday camp for cystic fibrosis patients. J. Clin. Microbiol. 33, 572–575 (1995). Kersulyte, D., Struelens, M. J., Deplano, A., Berg, D. E.: Comparison of arbitrarily primed PCR and macrorestriction (pulsed field gel electrophoresis) typing of Pseudomonas aeruginosa strains from cystic fibrosis patients. J. Clin. Microbiol. 33, 2216–2219 (1995). Mahenthiralingam, E., Campell, M. E., Foster, J., Lam, J. S., Speert, D. P.: Random amplified polymorphic DNA typ-
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ing of Pseudomonas aeruginosa isolates recovered from patients with cystic fibrosis. J. Clin. Microbiol. 34, 1129– 1135 (1996). National Committee for Clinical Laboratory Standards. Methods for diffusion antimicrobial susceptibility testing of aerobic bacteria, 4th edition. Approved standard. Publication M2-A6. National Committee for Clinical Laboratory Standards, Villanova, PA, USA (1997). Römling, U., Fiedler, B., Boßhammer, J., Gorthues, D., Greipel, J., von der Hardt, H., Tümmler, B.: Epidemiology of chronic Pseudomonas aeruginosa infections in cystic fibrosis. J. Infect. Dis. 161, 541–548 (1994). The International Pseudomonas aeruginosa Typing Study Group. A multicenter comparison of methods for typing strains of Pseudomonas aeruginosa predominantly from patients with cystic fibrosis. J. Infect. Dis. 169, 134–142 (1994). Wahba, A. H., Darrell, J. H.: The identification of typical strains of Pseudomonas aeruginosa. J. Gen. Microbiol. 38, 329–342 (1965).
Int. J. Med. Microbiol. 291, 387-393 (2001) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ijmm
Epidemiology of chronic Pseudomonas aeruginosa infections in cystic fibrosis Burçin S¸ener1, Özgen Köseog˘lu1, Ug˘ur Özçelik2, Tanıl Kocagöz1, Ayfer Günalp1 1 2
Hacettepe University, School of Medicine, Department of Microbiology and Clinical Microbiology, Ankara, Turkey Hacettepe University, School of Medicine, ˙I hsan Dog˘ramacı Children’s Hospital, Department of Chest Diseases, Ankara, Turkey
Received June 1, 2001 · Revision received July 26, 2001 · Accepted July 26, 01
Abstract Chronic lung infection with Pseudomonas aeruginosa is primarily responsible for pulmonary deterioration of cystic fibrosis patients. The purpose of this study was to type the P. aeruginosa isolates collected sequentially from cystic fibrosis patients, chronically colonized with P. aeruginosa, by random amplified polymorphic DNA fingerprinting-PCR (RAPD-PCR). Sequential P. aeruginosa isolates (n: 130) that had been collected from 20 CF patients over at least 9 years were investigated. The isolates were analyzed by RAPD-PCR using two arbitrary primers. Antimicrobial susceptibility testing of all isolates was performed by the disc diffusion method. RAPD-PCR typing demonstrated that strains dissimilar in colony morphotype and of different antibiotic susceptibility patterns could be of the same genotype. Some CF patients were colonized with a rather constant P. aeruginosa flora, with strains of different phenotypes but of one genotype. However, some patients may be colonized with more than one genotype. The results also demonstrated that there might be a risk of cross-colonization between CF patients followed-up at the same center. Key words: Cystic fibrosis – Pseudomonas aeruginosa – Random amplified polymorphic DNA fingerprinting (RAPD-PCR)
Introduction Chronic lung infection with Pseudomonas aeruginosa is primarily responsible for the pulmonary deterioration and reduced life expectancy in patients with cystic fibrosis (CF) (Gilligan, 1991; Gowan and Deretic, 1996). Once P. aeruginosa has taken residence in the CF lungs, it is rarely possible to eradicate it by antimicrobial chemotherapy (Gowan and Deretic, 1996). Analysis of such therapeutic failure requires distinction between persistence of the same strain with the same
susceptibility pattern, emergence of resistance in the same strain, or reinfection with a new strain. Due to the difficulties in typing isolates of P. aeruginosa from CF patients by conventional phenotypic typing techniques, the epidemiology of P. aeruginosa infections in CF patients is still not well defined. Genetic typing methods have shown to be more discriminatory than phenotypic methods for typing P. aeruginosa CF isolates (Grundmann et al., 1995; Mahenthiralingam et al., 1996; The International Pseudomonas aeruginosa Typing Study Group, 1994). Molecular
Corresponding author: B. Óener, Hacettepe University, School of Medicine, Department of Microbiology and Clinical Microbiologya, 06 100-Sıhhiye Ankara, Turkey, Phone: +90 312 3 051 560, Fax: +90 312 3 115 250, E-mail: [email protected] 1438-4221/01/291/5-387 $ 15.00/0
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typing of P. aeruginosa CF isolates revealed the persistence of single clones in some CF patients (Bingen et al., 1992; Fegan et al., 1991; Mahenthiralingam et al., 1996; Römling et al., 1994) and also co-colonization with one or more strains (Fegan et al., 1991; Mahenthiralingam et al., 1996). With the recent developments in genotyping techniques it became possible to reexamine the strains and to provide a definitive conclusion. PCR based fingerprinting (random amplified polymorphic DNA fingerprinting-PCR, RAPD-PCR) was applied in this study since it has already proven its value for the genotypic characterization of many medically important bacteria including P. aeruginosa (Belkum, 1994; Kersulyte et al., 1995). This study was conducted to type the P. aeruginosa isolates collected sequentially from 20 CF patients, to determine whether a single strain or multiple strains were responsible for chronic colonization and also to detect if there was any relation between the changes in the antimicrobial susceptibility pattern and the genotype of the isolates, and also to determine whether any cross-colonization/infection exists between the patients. We also tried to provide a conclusive answer for the risk of cross-infection between these CF patients.
Materials and methods Patients and isolates The epidemiology of chronic colonization of the airways with P. aeruginosa was studied for a 10-year period (1991– 2000), in 20 patients with CF attending Hacettepe University ˙I hsan Dog˘ramacı Children’s Hospital Paediatric Chest Diseases Unit. For each patient, the diagnosis of CF had been suspected by typical clinical findings of pulmonary and gastrointestinal disease and was subsequently confirmed by pilocarpine iontophoresis sweat tests. A total of 130 strains out of a total of 248 strains were included into the study. These 130 strains belonged to 20 patients who were selected out of 53 CF patients on the basis of colonization/infection for more than at least 6 months with P. aeruginosa. The remaining 33 patients had only 1 P. aeruginosa strain isolated during the study period. If the number of P. aeruginosa strains for a patient was more than 10, then 10 of these isolates were selected for genotype analysis according to the following criteria: 1) Simultaneous isolates of different colony morphotypes or antibiotic resistance patterns in one sputum sample, were taken as different strains. 2) Care was taken to select one strain as the representative of a certain morphotype or antibiotic resistance pattern during the follow-up for each patient. The follow-up periods of the selected cases, their age, sex characteristics, the colony morphotype changes, the changes in antibiotic resistance patterns, and the RAPD-PCR genotypes are shown in Table 1. P. aeruginosa strains were isolated from sputa or deepthroat swab specimens. Sputum cultures were performed at
regular intervals (monthly) for each patient (if the patient applied to the hospital for control) and also during infective exacerbations. The diagnosis of chronic P. aeruginosa infection was based upon the detection of P. aeruginosa in sputum for a period of at least 6 consecutive months. Isolates were identified as P. aeruginosa on the basis of typical morphology, positive oxidase reaction, ability to produce pigments, growth at 42 °C, and oxidative utilization of glucose (Gilligan, 1995). Colony morphologies were assessed as by Wahba and Darrell (1965), as mucoid, classic and rough. CF patients were called to the Chest Diseases Unit (an outpatient clinic) on the last Tuesday of each month for their routine control visits. These patients waited for 1–3 hours in the same waiting-room without wearing face-masks during these control visits. The approximate area of the waiting room is 15 m2 and the approximate distance of the patients varies between 1 to 5 m. The patients with or without P. aeruginosa infection/colonization were called for routine control visits without regarding P. aeruginosa infection. Environmental sampling was carried out from the single sink drain and the tap water in that room. The patients do not routinely wash their hands in this sink. All dry sites were sampled with sterile swabs moistened with sterile saline. The carriage of P. aeruginosa on staff hands was tested by taking samples from the staff examining these patients on that day. There was no instrument, or other area shared commonly by these patients. They used their own nebulizers for inhalatory treatment and the mouth applicator of the pulmonary function test equipment was disposable. There was no washbasin in the physiotherapy or pulmonary function test rooms. The staff working in those rooms washed their hands at the washbasin found in a separate room which was routinely tested by the Hospital Infection Control Committee. Antimicrobial susceptibility testing of all isolates was done by Kirby-Bauer disc diffusion method according to the guidelines of National Committee for Clinical Laboratory Standards (NCCLS) (1997). The tested antimicrobial agents were as follows: Aztreonam, piperacillin, ceftazidime, cefepime, sulbactam cefoperazone, meropenem, imipenem, ciprofloxacin, amikacin, tobramycin. The strains were stored at –70 °C until processed for RAPD-PCR analysis. Isolation of P. aeruginosa genomic DNA A single colony of P. aeruginosa was inoculated into 2 ml of trypticase soy broth and grown overnight at 37 °C. The bacteria were pelleted in a 1.5-ml microcentrifuge tube by spinning at 12 000 g for 3 minutes. The cells were then washed three times with 750 µl TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), boiled for 20 minutes in 500 µl TE, centrifuged, and the supernatant containing the DNA was stored at –20 °C until RAPD-PCR was applied. RAPD- PCR analysis Fourteen different primers (ECGA2: 5-TGCGCTGCCAGATGTCCGAG-3, SJMT3: 5-CCTGTGGGGTCCGGCCTTTC, ECGAr1: 5-GATAGAACCGAAGTTACCCT-3, T3: 5-CAAGGACGCACATGACAGGA, SJMT1: 5-ACCACC-
Epidemiology of Pseudomonas aeruginosa isolates
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Table 1. Summary of phenotypic and genotypic typing results of P. aeruginosa isolates derived from all CF patients studied. Patient
Sex
Age at the time of study (yr)
Time (month)
Total number of isolates tested
Colony morphology
Genotype
1
M
9
16
2
C
1
2
S
2
M
11
12
2
R
1
2
F
3
F
17
16
2
R
1
2
A
4
F
7
26
2
C
1
2
S
5
M
3
6
2
C
2
2
S
6
M
6
24
3
R
1
3
S, A
7
M
11
22
4
C
3
4
S
8
F
9
34
4
C
1
3 1
S, B
4
Number of isolates with genotype
Antibiotic resistance pattern
B
9
M
3*
28
5
C, M
1
5
S
10
M
7
25
5
R, C
5
6
4 1
S A
11
F
6
34
7
R, M
7, 7 8 9
4, 1 1 1
A F F
12
M
17*
57
10
C, M, R
10
10
S, A, G,H
13
M
21
40
10
C, M
11
8 2
S, A, C
14
M
20*
14
10
M
12
13
9 1
C, G, E E
15
M
18
72
10
M, R
14 15
8 2
S S
16
F
13
26
10
R, M
16
10
A, H
17
M
13
24
10
R, M
17
10
S
18
F
14
66
10
R, C
18
16
8 2
S, B S
10
C
19
M
15
36
11
M, R
19 20 21
9 1 1
S, F S H
20
F
10
24
11
C, M, R
22 23 24
9 1 1
S S S
Colony morphology; M: Mucoid, C: Classic, R: Rough. *; Exitus.
Antibiotic resistance patterns; S: Susceptible to all antibiotics tested, A: Resistant to ceftazidime, aztreonam, B: Resistant to amikacin and/or tobramycin, C: Resistant to ceftazidime, amikacin, D: Resistant to ceftazidime, amikacin, ciprofloxacin, E: Resistant to ceftazidime, amikacin, ciprofloxacin, imipenem, F: Resistant to tobramycin, imipenem, G: Resistant to ceftazidime, aztreonam, imipenem, H: Resistant to ceftazidime, aztreonam, imipenem and amikacin.
GAGCGGTTCGCCTGA-3, M1: 5-GAAGCTTATGGTACAGGTTGG, TT15: 5-TCGCGAATCAGCTCGCCG-3, SJMTr2: 5-GATCTGCGGGTCGTCCCAGGT-3, MT2: 5-CTCGTCCAGCGCCGCTTCGG-3, MTBGyrAend: 5TCACCCCGACTCCTAACACT-3, INHA1: 5-CCACAACTAGAATGCAGTGAAAAAA, M2: 5-ATTACCATCCTTGTTGTAAG, L2: 5-CTGGCTTCTTCCAGCTTCA-3, TT16: 5-GCTTTGCAAGCTCCTCACC-3) of 18–26 nucleotides, used for amplification of various genes of several other microorganisms, were initially screened for reproduc-
ibility and the ability to produce discriminatory polymorphisms by using DNA of P. aeruginosa ATCC 27 853. Four primers (SJMT3, M1, T3 and INHA1) were found to result in reproducible and discriminatory amplification of DNA fingerprints; primer SJMT3 was selected for primary typing of the CF isolates described in this study, and primer M1 was used for confirmation. SJMT3 and M1 were selected upon their higher GC content. Each strain was typed twice under the same conditions to test reproducibility and reliability of the results.
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RAPD-PCR mixtures of 25 µl, containing 1 reaction buffer, 2.5 mM MgCl2, 0.2 mM (each) deoxynucleosidetriphosphates (dNTP), 0.65 U Replitherm polymerase (Epicentre Technologies), 30 pM primer and 2 µl sample DNA were prepared. The DNA was denatured for 3 min at 94 °C. At the beginning, 3 cycles were performed with an automated thermal cycler (MJR Research) at low annealing temperature. Each cycle consisted of denaturation at 94 °C for 1 min, annealing of primers at 30 °C for 1 min and primer extension at 72 °C for 1 min. Then 45 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min, followed by a final extension step at 72 °C for 3 min were performed. RAPD-PCR products (10 ml of each reaction mixture) were then separated by electrophoresis in 1.5 % agarose gels with 1 TAE running buffer. Gels were stained with ethidium bromide and visualized on a UV transilluminator and photographed. The bands were further examined by Ultra Violet Products (UVP) White/UV transilluminator and GrabIT annotating grabber program. Gel images were then analyzed by GelWorks ID Intermediate Version 2.51 (Nonlinear Dynamics Ltd) according to the manufacturer’s instructions.
First, sample and molecular weight marker lanes were located manually. After automatic band detection, a visual inspection was carried out in order to determine the unrecognized bands. For each gel, φx174 was used as molecular weight standard to calculate the absolute lengths of the bands in individual sample lanes. Those calculations in turn, yielded numeric band patterns that are suitable for the evaluation of each strain. Each unique band pattern was denoted with letters from “1 to 24”. Very closely related genotypes in which only sub-band differences were found were coded by the same number with an additive (7 versus 7).
Results The 20 patients taken into the study yielded a total of 248 P. aeruginosa strains and among these 130 strains were genotyped according to the criteria mentioned above. The results of environmental and clinical staff screening performed by us and by the routine Hospital Infection Control Committee revealed no P. aeruginosa isolation. Selection of primers for RAPD-PCR analysis of P. aeruginosa
Fig. 1. P. aeruginosa ATCC 27853 strain-specific DNA polymorphisms generated by 14 different AP-PCR primers. Molecular size markers were run in lane M and their sizes (in base pairs) are as follows: 1353, 1078, 872, 603, 310, 281. The primers tested were as follows, in order of the lanes above (1– 14): ECGA2, SJMT3, ECGAr1, T3, SJMT1, M1, TT15, SJMTr2, MT2, MTBGyrAend, INHA1, M2, L2, TT16.
Fig. 2. The common RAPD genotype 1, shared by the patients (6, 2, 3, 4, 9, 1 and 8), generated by the primer M1. The first lane denoted by “M” is the molecular weight marker φx174.
Among the 14 primers tested, 4 were found to result in reproducible and discriminatory amplification polymorphisms suitable for strain differentiation of P. aeruginosa. The P. aeruginosa strain-specific polymorphisms generated by different RAPD-PCR primers are shown in Figure 1. The suitable primers were all of high GC content. SJMT3 and M1 were selected since they produced more bands than the other primers tested. Each primer yielded RAPD-PCR patterns which enabled the differentiation of unique strains of P. aeruginosa. The RAPD-PCR patterns revealing bands with different numbers, sizes and intensities were considered to represent different isolates. RAPD-PCR amplification patterns obtained from the 20 patients revealed 24 unique RAPD types (Table 1). Seven patients (patients 1, 2, 3, 4, 6, 8, 9) shared the same genotype, denoted by 1, the strains being isolated between 1992–2000, and possibly originated from patient 9 (strain first isolated in May 1992) (Figure 2). Of these seven patients, six patients (patients 1, 3, 4, 5, 6 and 9) harbored this genotype consistently within their lungs during repeated sampling over a 12months period. However, patient 8 was found to harbor a second genotype in only one sample besides the common shared one. Besides these 7 patients, patients 12 and 13, and patients 16 and 18 shared common genotypes, genotypes 10 and 16, respectively. Fifteen patients had P. aeruginosa isolates with one to three different genotypes. Among the 20 patients, 13 exhibited
Epidemiology of Pseudomonas aeruginosa isolates
a single persistent colonizing genotype. A “persistent colonizing genotype” was defined as the same genotype isolated from all specimens (at least 5 specimens) of a patient collected over the study period. “Transient colonizing genotypes” were defined as those isolated only from one or two of the specimens collected during the study period, suggesting reinfection, or colonization. Nine patients (patients 8, 10, 11, 13, 14, 15, 18, 19, 20) showed transient colonization with genotypes other than the persistent genotype. For example for the patient 11, the RAPD genotype of the strains collected between January 1997 and October 2000 are given in Figure 3, lanes 4 and 5 reveal the genotypes (7 and 7) of strains simultaneously isolated in the same sputum sample, with different colony morphotypes. Lanes 6 and 7 denote strains isolated in June 1999 (genotype 8) and October 2000 (genotype 9), respectively. Lane 8 belongs to genotype 7, which was the initial persistent genotype for that patient. The results revealed that the genotype 7 was the persistent genotype while the others (7, 8 and 9) represent transient colonization. Nine patients harbored more than one genotype during their whole follow-up and sometimes simultaneously i. e. two different patterns could be found in one sputum sample, e. g. patients 13, 15 and 18. The genotyping of sequential isolates from the nine patients, 12 through 20, revealed that each of these patients was chronically colonized/infected with strains of a single predominating RAPD type which remained stable for isolates collected over several years. For example, the first and the last isolates of patient 12 (RAPD type 10) were recovered 4.7 years apart, isolates from patient 15 (RAPD type 14) 6 years apart, isolates from patient 17 (RAPD type 17) 3 years apart (Figure 4). Six of these 9 patients were co-colonized at one time or another with two or more strains of different RAPD type, e. g. patient 19 was found to harbor isolates with RAPD types 19 and 20 at one visit. Many isolates identified as identical by RAPD genotype displayed a variable colony morphotype. For example isolates from patient 12 with RAPD type 10 exhibited classic, mucoid and rough colony forms at different isolation dates. Genotypically identical isolates also exhibited variability in antibiotic resistance pattern. For example, the isolates in RAPD type 10 for patient 12, RAPD type 11 for patient 13, RAPD type 12 for patient 14, RAPD type 18 for patient 18, RAPD type 19 for patient 19 revealed different antibiotic resistance profiles.
Discussion The poor prognosis of CF is especially associated with an early onset of P. aeruginosa lung infection. Typing
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Fig. 3. RAPD-PCR profile of the patient 11, generated by the primer M1. The first lane denoted by “M” is the molecular weight marker φx174. The strains were collected between January 1997 and October 2000. Lanes 4 and 5 reveal the genotypes (7 and 7) of strains simultaneously isolated in the same sputum sample, with different colony morphotypes. Lanes 6 and 7 denote strains isolated in June 1999 and October 2000, respectively.
Fig. 4. RAPD-PCR profile of patient 17, generated by the primer M1.The first lane denoted by “M” is the molecular weight marker φx174. The 10 sequential isolates collected between November 1996 and December 1999 show the same band pattern.
of P. aeruginosa isolates of CF origin by conventional phenotypic methods has been found to be difficult due to either the mucoid nature or the rough outer membrane of the isolates. Detailed analysis of P. aeruginosa populations in chronically infected CF patients is necessary, in particular to examine the complexity and stability of bacterial populations and to understand better how they adapt and evolve. More recently, genetic methods have been applied to study the epidemiology of P. aeruginosa in CF patients (Grundmann et al., 1995; The International Pseudomonas aeruginosa Typing Study Group, 1994). Of the many DNA-based typing methods, RAPD-PCR seemed to be an efficient and sensitive means of high-throughput typing of such strains (Belkum, 1994; Hoogkamp-Korstanje et al., 1995; Kersulyte et al., 1995). This longitudinal study revealed that most CF patients were harboring the same P. aeruginosa genotype
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from the onset of colonization throughout the whole follow-up period. Turnover of the predominant genotype was not seen in any of the cases. This result was also obtained in some other previous studies (Kersulyte et al., 1995; Römling et al., 1994). However, occasional transient, or consistent genotypes were recovered in some of the patients (patients 8, 10, 11, 13, 14, 15, 18, 19, 20), usually simultaneously with the recovery of the predominant genotype. Some researchers contributed the absence of any predominant genotype to the limited time course of the study (Fegan et al., 1991). This was not the case in this study, since most patients were followed-up for more than 24 months. In general, despite alterations in the colony morphotype and antibiotic resistance profile, the RAPD fingerprints of sequential isolates remained stable, suggesting that these colonizing isolates enhanced their capacity to survive in the lower respiratory tract of CF patients. The question of cross-colonization in CF patients has not been conclusively resolved (HoogkampKorstanje et al., 1995). The results of this study suggest that there is risk of cross-colonization between CF patients followed-up at the same CF center. It was observed that RAPD-type 1 was the most commonly shared P. aeruginosa genotype in this study population, being recovered in 7 patients. It is noteworthy that P. aeruginosa RAPD type 1 was first isolated in May 1992 in patient 9 and was still present in the isolates of some of the patients in 2000. The risk of crosscolonization in our CF center may be due to the fact that CF patients are called for routine follow-up to the center on every last Tuesday of the month, and are educated for physiotherapy at the same place. Since these patients used their own nebulizers for treatment and no P. aeruginosa was isolated from the hands of staff members, this route of transmission does not seem to be a major factor for cross-colonization in these cases. None of these patients were known to socialize together outside the hospital, and none of them were siblings. It had been shown by Doring et al. (1991) that aerosols containing P. aeruginosa are known to be generated during hand washing from colonized or infected patients. During this study, although no P. aeruginosa has been isolated from washbasins and tap water, the U-shaped tube of these sinks was not sampled. Therefore, since there might be a P. aeruginosa source in these tubes, according to our results it can be concluded that this route plus being together in a small, crowded room with cough and sneeze droplets seemed to be the only possible ways, although further studies to ascertain the exact mode of transmission of P. aeruginosa between CF cases seem to be necessary. It is a matter of debate why other CF patients in our center are not colonized with RAPD type 1. An explanation for this may be that the other patients’ initial coloniz-
ing genotypes may outcompete this common genotype within the lung environment. Like other investigators, we have found that molecular characterization is the most helpful tool for typing CF P. aeruginosa strains. We used RAPD-PCR because it has high discriminatory power and already has proven its value in epidemiological studies of medically important microorganisms including P. aeruginosa (Belkum, 1994). Data presented here show that some of the CF patients are colonized with a rather constant P. aeruginosa flora, with strains of different phenotypes but of one genotype over a long period of time. However, we also found patients colonized with more than one genotype simultaneously, suggesting that the composition of the flora is not constant as a rule, but it may fluctuate considerably. These results also suggest the presence of risk of cross-colonization among our CF patients, and as a precaution these patients are now being called for control visits on different days of the month and are told to wear face masks in the waiting room. Acknowledgement. We thank Dr. Doruk Engin for his technical help in analysis of gel images. This study was partly presented at the 22nd European CSConference, June 13–19 1998, Berlin.
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