Abstract
A method for distinguishing among Pseudomonas aeruginosa strains using random amplified polymorphic DNA (RAPD) typing was evaluated for reproducibility and discriminatory power. A total of 200 isolates, blinded in triplicate, were evaluated by RAPD. All 600 samples were typeable; 197 of 200 isolates gave identical results on all three occasions, and 131 distinct RAPD types were identified.
Pseudomonas aeruginosa is the predominant respiratory pathogen in patients with cystic fibrosis (CF) (11), but its means of acquisition and the risk of patient-to-patient spread remain poorly understood. To enable epidemiological studies of sequential isolates from CF patients, a reliable and simple method of fingerprinting is necessary. This study describes the evaluation of a method for typing P. aeruginosa using random amplified polymorphic DNA (RAPD) fingerprinting.
Bacterial isolates.
A total of 200 isolates of P. aeruginosa, obtained from a previous study (4), were frozen in triplicate (600 samples), each with a unique blinded code number. The sources included 150 isolates from 109 patients with CF (from five geographically distinct locations), 17 International Antigenic Typing System (IATS) type strains, 23 isolates from 22 non-CF patients, and 10 isolates from the environment (river and vegetables).
RAPD typing.
Samples were grown from frozen stock on Columbia agar with 5% sheep's blood (Prepared Media Laboratories, Richmond, British Columbia, Canada). Pure cultures were prepared for RAPD analysis as previously described (7), and extracted DNA was amplified using sequence 272 (5′-AGCGGGCCAA-3′) and sequence 208 (5′-ACGGCCGACC-3′). RAPD patterns were analyzed by an observer who was blind to isolate identity; comparisons among and between samples were performed both visually and using Molecular Analyst Fingerprinting (MAF) software (Bio-Rad Laboratories) with Pearson product-moment correlation coefficient, unweighted pair group method using arithmetic averages, and global optimization. Banding patterns with a similarity index of >75% by MAF or which appeared similar visually were run again, side by side on the same gel, to confirm the identity. Two or more samples with the same banding pattern were assigned a number indicating a single RAPD type. Those with no more than one major band or three minor band differences were thought to be possibly related and subjected to further analysis by pulsed-field gel electrophoresis (PFGE) (6).
Typeability and reproducibility.
Of 200 isolates, 197 yielded reproducible fingerprints from all three samples by RAPD analysis (i.e., the same RAPD type) (Table 1). The remaining three isolates yielded reproducible results in two of three samples. A total of 150 isolates from 109 CF patients from six clinics were analyzed. Multiple morphotypes (two to four) were evaluated from each of 32 patients; in all cases, the different samples from each patient yielded identical RAPD patterns.
TABLE 1.
Typeability and reproducibility of RAPD analysis of P. aeruginosa isolatesa
| Source of P. aeruginosa | No. of isolates tested | No. of isolates (%) demonstrating:
|
||
|---|---|---|---|---|
| Typeable in triplicate | Reproducible in triplicate | Reproducible in duplicate | ||
| CF patients | 150 | 150 (100) | 149 (99.3) | 1 (0.7) |
| Non-CF patients (clinical) | 23 | 23 (100) | 22 (95.7) | 1 (4.3) |
| Environmental | 10 | 10 (100) | 9 (90.0) | 1 (10.0) |
| IATS | 17 | 17 (100) | 17 (100) | 0 |
| Total | 200 | 200 (100) | 197 (98.5) | 3 (1.5) |
A total of 200 isolates were typed in triplicate by RAPD and compared as described in the text. The typing reaction was considered reproducible if it yielded an identical pattern on two or more occasions.
RAPD types.
There were a total of 131 RAPD types identified among the 600 samples (Table 2). Of 38 types, each contained more than one isolate, 15 of which were multiple isolates from individual patients. A total of 108 RAPD types were unique to a single patient or source (Table 2), including 66 types unique to CF patients (80 of 150 isolates) and 16 types unique to non-CF patients. All 10 environmental and 16 of 17 IATS isolates yielded unique RAPD types. In total, 23 RAPD types were shared by multiple patients or sources (Table 2), including 4 types by sibling pairs, 14 types by unrelated CF patients, 3 types by CF with non-CF patients, 1 type by unrelated non-CF patients, and 1 type by a non-CF patient with an IATS strain.
TABLE 2.
Distribution of unique and shared RAPD types among different sources of P. aeruginosa
| Source of P. aeruginosa | No. of isolates tested | No. of sources (type) | No. of unique types | No. of shared types (shared with) |
|---|---|---|---|---|
| CF patients | 150 | 109 (patients) | 66 | 4 (sibling pairs only) |
| 14 (unrelated CF patients) | ||||
| 2 (CF and non-CF patients)a | ||||
| 1 (CF and non-CF patients and sibling pair)a | ||||
| Non-CF patients | 23 | 22 (patients) | 16 | 1 (unrelated non-CF patients) |
| 2 (CF and non-CF patients)a | ||||
| 1 (CF and non-CF patients and sibling pair)a | ||||
| 1 (non-CF patients and IATS)a | ||||
| Environmental | 10 | 6 (vegetables) | 10 | 0 |
| IATS | 17 | 17 (patients) | 16 | 1 (non-CF patients and IATS)a |
| Total | 200 | 158 | 108 | 23a |
RAPD types shared between different sources are included only once in the total.
The epidemiology of P. aeruginosa in CF patients is controversial. Some studies have suggested patient-to-patient spread (1–3, 10), whereas others have failed to find such evidence (5, 8, 12, 13). It is important to clarify the epidemiology of P. aeruginosa in CF in order to rationally design infection control policies. A previous study (4) indicated that restriction fragment length polymorphism analysis using a probe upstream from the gene for exotoxin A proved to have the greatest discriminatory power. This approach was significantly superior to phenotypic methods, yielding the greatest number of distinct patterns among isolates that were reproducibly typed (54 unique types among 145 isolates). This method indicated that the changes in colonial morphology of sequential isolates from a patient do not represent replacement by a new strain but rather phenotypic plasticity (7, 9). Before evaluation of a large number of patient isolates from Canadian clinics to further clarify the epidemiology of infection in CF, it was necessary to first determine if RAPD typing (a simple gene-based technique) could give reproducible and discriminatory results.
The study employed the same 600 samples from the previous investigation (4) to evaluate RAPD typing and found that it had a high degree of reproducibility, with 98.5% yielding the same banding patterns for each triplicate. Epidemiologically related isolates, such as multiple isolates from individual patients and those from each of five sibling pairs, were appropriately “read” as identical by RAPD. Isolates that were epidemiologically unrelated, such as those from the environmental and the IATS type strains, each had a unique RAPD pattern.
This method was highly reproducible and was able to differentiate apparently unrelated strains. RAPD typing is a robust, simple, and highly reproducible method that should be useful in clarifying the epidemiology of P. aeruginosa in patients with CF. RAPD typing is best suited to situations in which a large number of isolates are to be evaluated. Results from RAPD analysis can be used in conjunction with the more cumbersome PFGE method to resolve and confirm the difference or identity among smaller numbers of isolates when an ambiguous result is obtained with RAPD.
Acknowledgments
This work was supported by a grant to D.P.S. from the Canadian Cystic Fibrosis Foundation.
We thank Spencer Matheson, Nicole Glenham, and Gary Probe for excellent technical support.
REFERENCES
- 1.Cheng C K, Smyth R L, Govan R, Doherty C, Winstanley C, Denning N, Heaf D P, van Saene H. Spread of beta-lactam-resistant Pseudomonas aeruginosa in a cystic fibrosis clinic. Lancet. 1996;348:1596–1597. doi: 10.1016/S0140-6736(96)05169-0. [DOI] [PubMed] [Google Scholar]
- 2.Farrell P M, Shen G, Splaingard M, Colby C E, Laxova A, Kosorok M R, Rock M J, Mischler E H. Acquisition of Pseudomonas aeruginosa in children with cystic fibrosis. Pediatrics. 1997;100:E2. doi: 10.1542/peds.100.5.e2. [DOI] [PubMed] [Google Scholar]
- 3.Høiby N, Rosendal K. Epidemiology of Pseudomonas aeruginosa infection in patients treated at a cystic fibrosis centre. Acta Pathol Microbiol Scand B. 1980;88:125–131. doi: 10.1111/j.1699-0463.1980.tb02617.x. [DOI] [PubMed] [Google Scholar]
- 4.International P. aeruginosa Typing Study Group. A multicenter comparison of methods for typing strains of Pseudomonas aeruginosa from patients with cystic fibrosis. J Infect Dis. 1994;169:134–142. doi: 10.1093/infdis/169.1.134. [DOI] [PubMed] [Google Scholar]
- 5.Kelly N M, Fitzgerald M X, Tempany E, O'Boyle C, Falkiner F R, Keane C T. Does Pseudomonas cross-infection occur between cystic fibrosis patients? Lancet. 1982;ii:688–690. doi: 10.1016/s0140-6736(82)90714-0. [DOI] [PubMed] [Google Scholar]
- 6.Lightfoot J, Lam J S. Chromosomal mapping, expression and synthesis of lipopolysaccharide in Pseudomonas aeruginosa: a role for guanosine diphospho (GDP)-d-mannose. Mol Microbiol. 1993;8:771–782. doi: 10.1111/j.1365-2958.1993.tb01620.x. [DOI] [PubMed] [Google Scholar]
- 7.Mahenthiralingam E, Campbell M E, Foster J, Lam J, Speert D P. Random amplified polymorphic DNA typing of Pseudomonas aeruginosa isolates recovered from patients with cystic fibrosis. J Clin Microbiol. 1996;34:1129–1135. doi: 10.1128/jcm.34.5.1129-1135.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Ogle J W, Janda J M, Woods D E, Vasil M L. Characterization and use of a DNA probe as an epidemiological marker for Pseudomonas aeruginosa. J Infect Dis. 1987;155:119–126. doi: 10.1093/infdis/155.1.119. [DOI] [PubMed] [Google Scholar]
- 9.Pasloske B L, Joffe A M, Sun Q, Volpel K, Paranchych W, Eftekhar W, F, Speert D P. Serial isolates of Pseudomonas aeruginosa from a cystic fibrosis patient have identical pilin sequences. Infect Immun. 1988;56:665–672. doi: 10.1128/iai.56.3.665-672.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Pedersen S S, Koch C, Høiby N, Rosendal K. An epidemic spread of multiresistant Pseudomonas aeruginosa in a cystic fibrosis centre. J Antimicrob Chemother. 1986;17:505–516. doi: 10.1093/jac/17.4.505. [DOI] [PubMed] [Google Scholar]
- 11.Speert D P. Pseudomonas aeruginosa infections in patients with cystic fibrosis. In: Baltch A L, Smith R P, editors. Pseudomonas aeruginosa infections and treatment. New York, N.Y: Marcel Decker; 1994. pp. 183–234. [Google Scholar]
- 12.Speert D P, Campbell M E. Hospital epidemiology of Pseudomonas aeruginosa from patients with cystic fibrosis. J Hosp Infect. 1987;9:11–21. doi: 10.1016/0195-6701(87)90089-2. [DOI] [PubMed] [Google Scholar]
- 13.Speert D P, Lawton D, Damm S. Communicability of Pseudomonas aeruginosa in a cystic fibrosis summer camp. J Pediatr. 1982;101:227–229. doi: 10.1016/s0022-3476(82)80127-3. [DOI] [PubMed] [Google Scholar]