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Pseudomonas aeruginosa and other gram-negative nonfermenting bacilli such as Burkholderia cepacia, Stenotrophomonas maltophilia, and Alcaligenes xylosoxidans are a major cause of chronic lung infection in patients with cystic fibrosis (CF) (5). It is essential to identify these organisms accurately to species level because they differ in clinical significance. P. aeruginosa is associated with progressive pulmonary deterioration and poor clinical prognosis (9, 10). Its isolation from CF patients is therefore a major prognostic indicator. The isolation of B. cepacia from CF patients also has major medical and infection control implications because this organism may cause rapidly fatal, necrotizing pneumonia (18) and is resistant to multiple antibiotics (12) and at least some of its clones spread efficiently from patient to patient in hospitals and via social contact (13, 16, 17). In contrast, S. maltophilia and A. xylosoxidans are mostly isolated after lengthy colonization by P. aeruginosa and are generally considered to be harmless colonizers selected after repeated or prolonged treatment for P. aeruginosa infection (3, 5).

The phenotypic identification of P. aeruginosa and other gram-negative nonfermenting bacilli recovered from chronically colonized CF patients is difficult. A large proportion of strains are atypical in terms of the appearance of colonies, metabolism, antibiotic susceptibility, and immunoreactivity (4, 7, 14, 15). Several commercial kits are available for the identification of gram-negative nonfermenting bacilli. Unfortunately, the overall accuracy of these systems is poor for strains isolated from CF patients. A recent study found that the accuracy of four commercial systems for identifying 150 clinical isolates of nonfermenting gram-negative bacilli recovered from CF patients was only 57 to 80% (11). Therefore, the identification of gram-negative nonfermenting bacilli recovered from CF patients is still based on conventional biochemical testing and remains a fastidious, difficult, and time-consuming procedure. Even this approach is not totally reliable. A recent paper reported the misidentification of S. maltophilia as B. cepacia by diagnostic laboratories with experience in working with organisms isolated from CF patients (2).

Genotypic identification methods overcome these problems. A new approach based on single-strand conformation polymorphism (SSCP) analysis of PCR-amplified 16S rRNA gene fragments has been developed (20, 21). SSCP patterns were initially analyzed by nondenaturing polyacrylamide gel electrophoresis and silver staining (20). This protocol was then modified to include fluorescence-based PCR-SSCP coupled to an automated DNA sequencer (21). A single nucleotide difference in the amplified region was sufficient to obtain different patterns with fluorescence-based SSCP analysis. This made it possible to identify a broad range of gram-positive and gram-negative bacteria accurately, including P. aeruginosa and S. maltophilia (21). Capillary electrophoresis (CE) is a new electrophoretic technique in which slab gels are replaced by capillaries. CE-SSCP analysis is particularly useful for the detection of point mutations associated with inherited diseases (1, 8). In this study, we used CE-SSCP analysis for the rapid identification of P. aeruginosa and other nonfermenting gram-negative bacilli recovered from CF patients.

Test organisms. The strains used in this study were 34 reference strains (Table 1) and 207 clinical isolates of gram-negative nonfermenting bacilli (gram-negative, rod-shaped, cytochrome c oxidase-positive organisms utilizing glucose oxidatively) recovered from the respiratory tracts of CF patients. Clinical isolates were obtained from the frozen strain collections of the microbiology laboratories of Necker-Enfants Malades Hospital and Robert Debré Hospital, Paris, France, and Centre Hospitalier Régional et Universitaire, Lille, France. They included 153 P. aeruginosa, 26 S. maltophilia, 11 B. cepacia, 4 Burkholderia gladioli, and 13 A. xylosoxidans isolates. Multiple isolates of the same species from an individual patient were not included, except for P. aeruginosa isolates with colonies differing in appearance (e.g., fried-egg pigmented and mucoid nonpigmented P. aeruginosa). Clinical isolates were identified by standard methods (6, 19). P. aeruginosa isolates were serotyped by the slide agglutination technique, with commercially prepared antisera against 16 somatic O antigens (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France). Strains that were not agglutinated or were agglutinated by more than one typing serum were classed as nontypeable. The mucoid aspect of the colonies was also noted.

DNA amplification. Bacterial strains were cultured overnight at 35°C on tryptic soy agar, scraped from the plates, and suspended in distilled water to give an optical density of 1.2 to 1.3 at 600 nm. Bacterial cells were lysed by heating at 100°C for 10 min. They were centrifuged for 2 min, and 5 µl of the supernatant was then directly used for PCR. The PCR mixture (final volume = 100 µl) contained 0.1 µM (each) primer, 200 µM (each) deoxynucleoside triphosphates, and 2.5 U of Taq DNA polymerase (ATGC Biotechnologies, Noisy-le-Grand, France), in 1× amplification buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl₂). Three sets of primers, purchased from GENSET SA (Paris, France), were used for amplification of the 16S rRNA gene regions. The first set of primers was P11P (5'-GAG GAA GGT GGG GAT GAC GT) and P13P (5'-AGG CCC GGG AAC GTA TTC AC), to amplify a 217-bp fragment (Escherichia coli 16S rRNA positions 1173 to 1389) (20, 21). The second set of primers was ER14 (5'-GCT AAC TCC GTG CCA GCA) and ER15 (5'-GCG TGG ACT ACC AGG GTA TC), to amplify a 305-bp fragment (E. coli 16S rRNA positions 506 to 810) (21). The third set of primers (this study) was MM3 (5'-GCA GCA GTG GGG AAT TTT GG) and MM4 (5'-TTA CGC CCA GTA ATT CCG AT), to amplify a 213-bp fragment (E. coli 16S rRNA positions 359 to 571). The forward primers (P11P, ER10, and MM3) were labeled with the fluorescent dye TET (green), and the reverse primers (P13P, ER11, and MM4) were labeled with the fluorescent dye FAM (blue). The PCR was performed with 25 cycles of 1 min at 94°C, 10 s at 72°C, and 1 min at 55°C. Five microliters of the amplified product was loaded onto an agarose gel and subjected to electrophoresis, and the gel was viewed under UV light to check for DNA amplification.

CE-SSCP analysis. CE-SSCP electrophoresis was performed on an ABI PRISM 310 genetic analyzer (Perkin-Elmer). Experimental conditions were optimized by using P11P-P13P PCR products from P. aeruginosa ATCC 10145^T. We evaluated 2 to 4% polymer and 5 to 10% glycerol. The best results were obtained with 4% polymer and 10% glycerol. Perkin-Elmer recommends purifying PCR products by removing salts and excess primers with Amicon Centricon-100 columns (Millipore, Bedford, Mass.). In our hands, this step did not appear to be absolutely necessary, providing that the PCR products were used at dilutions greater than 1/50 for SSCP analysis. Perkin-Elmer also recommends improving DNA heat denaturation by using both formamide and NaOH. Preliminary trials showed that adding NaOH did not improve DNA denaturation and that it affected the mobility of single-stranded DNA molecules. DNA was therefore heat denatured in the presence of formamide alone in our final protocol. The deionization of formamide was essential and was carefully performed. The definitive protocol used with P11P-P13P was as follows. After thermal cycling, 1 µl of the PCR mixture diluted 1:100 was added to 14 µl of sampling buffer (12.5 µl of deionized formamide, 1.0 µl of distilled water, and 0.5 µl of GeneScan 500 standard). GeneScan 500 standard was labeled with the fluorescent dye TAMRA (yellow). Formamide was deionized by using Ag501-X8 resin (Bio-Rad Laboratories, Hercules, Calif.). The sample was heated for 3 min at 95°C and chilled on ice before being loaded on the analyzer. The SSCP analysis gel contained 4% GeneScan polymer, 10% glycerol, and 1× Tris-borate-EDTA. The electrophoresis conditions were as follows: capillary, Lt, 41 cm, and Ld, 30 cm; temperature, 30°C; electric field, 317 V/cm. SSCP patterns were analyzed with GeneScan 672 software (Perkin-Elmer). The conditions established with P11P-P13P were applied to ER10-ER11 and MM3-MM4. Similar results were obtained, but PCR products were diluted only 1/10 for CE-SSCP analysis because the yield of PCRs was lower with these primer sets. Purification of PCR products with Amicon Centricon-100 columns was therefore required. The protocol was otherwise the same as that with P11P-P13P.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Correction of lane-to-lane variations with GeneScan 672. Curves: A, internal control (GeneScan 500 standard); B, electropherograms before alignment; C, electropherograms after alignment. Curves B and C consist of the five electropherograms obtained with the same sample passed five times in the same run, superimposed on one another. x axis, scan values; y axis; fluorescence intensity. Before alignment (B), a shift is clearly apparent between samples; after alignment (C), the samples have similar migration patterns.

CE-SSCP analysis of reference strains. (i) Primer set P11P-P13P. PCR products obtained from reference strains with the primer set P11P-P13P were analyzed by CE-SSCP. We first tested the main species of the genera Pseudomonas, Brevundimonas, Burkholderia, Comamonas, Stenotrophomonas, and Alcaligenes: P. aeruginosa, Brevundimonas vesicularis, B. cepacia, Comamonas acidovorans, S. maltophilia, and A. xylosoxidans, respectively. A specific pattern was observed for each of the six species tested (Fig. 2). Both forward and reverse strands underwent mobility shifts and were useful for discrimination between species. In addition to the strain ATCC 10145^T, 11 reference P. aeruginosa strains belonging to various serotypes were studied. All strains tested gave the same pattern (data not shown), confirming the reliability of CE-SSCP analysis. The discriminatory power of P11P-P13P for closely related species was evaluated (Table 2). Among Pseudomonas species, P. aeruginosa, Pseudomonas alcaligenes, and Pseudomonas pseudoalcaligenes gave patterns that were indistinguishable from each other. The pattern of Pseudomonas stutzeri was related but significantly different (mobility values for the forward strand differing by four scan numbers). The same pattern was observed for Pseudomonas fluorescens and Pseudomonas putida, but this pattern was clearly different from that of P. aeruginosa (Fig. 2). This was not very surprising, because P. fluorescens, P. putida, and P. aeruginosa are classed together in the fluorescent pseudomonad group but P. fluorescens and P. putida differ substantially from P. aeruginosa in the nucleotide sequence of 16S rRNA. The patterns of Brevundimonas diminuta and B. vesicularis were similar and were distinct from other patterns observed for other genera. The range of mobility values was broader with the reverse strand (scan numbers of 3914 to 3928 versus 3825 to 3830 with the forward strand). However, both strands were informative. For example, only the forward strand differentiated P. aeruginosa from P. stutzeri. Differences in mobility values were limited among species of the genus Burkholderia. However, B. cepacia gave a pattern that was different from the others. The patterns of Burkholderia cocovenenans, B. gladioli pv. alliicola, B. gladioli pv. gladioli, Burkholderia glumae, and Burkholderia plantarii could not be distinguished from one another. Finally, P11P-P13P could not discriminate between Alcaligenes species: the mobility values for the forward strand were similar for the four species tested, and those for the reverse strand differed by only two scan numbers.

View larger version (28K): [in this window] [in a new window]   FIG. 2.   CE-SSCP analysis of reference strains with P11P-P13P. Curves: A, P. aeruginosa ATCC 10145T; B, B. cepacia CIP 80.24T; C, C. acidovorans ATCC 15668T; D, B. vesicularis ATCC 11426T; E, S. maltophilia CIP 60.77T; F, A. xylosoxidans CIP 61.20. x axis, scan values; y axis, fluorescence intensity. Samples were analyzed in the same run and aligned by using the GeneScan 500 standard.

(ii) Primer sets ER14-ER15 and MM3-MM4. The ER14-ER15 primer set was used to identify Pseudomonas species. Distinct patterns were obtained with all Pseudomonas species tested. However, the major peaks each consisted of two to three peaks, the relative importances of which differed from one PCR to another (data not shown). We tried without success to overcome this by changing the experimental conditions. The MM3-MM4 primer set was used for the identification of Burkholderia and Alcaligenes species. The discrimination of Burkholderia species was no better than that with P11P-P13P (Table 3). For example, the MM3-MM4 set did not differentiate among B. cocovenenans, B. gladioli pv. alliicola, B. glumae, and B. plantarii. The use of MM3-MM4 was also less effective than was hoped for in identifying Alcaligenes species. In particular, this set of primers did not differentiate between A. xylosoxidans and Alcaligenes denitrificans (Table 3).

CE-SSCP analysis of CF isolates. PCR products from 207 clinical isolates were tested with the P11P-P13P primer set. P. aeruginosa isolates included 47 typeable; 48 nonmucoid, nontypeable; and 58 mucoid, nontypeable isolates. Other isolates included 26 of S. maltophilia, 11 of B. cepacia, 4 of B. gladioli, and 13 of A. xylosoxidans. Control injections were made every 20th injection (three injection controls for a 48-well-plate) to overcome the problem of differences between lanes. Controls included the reference strains P. aeruginosa ATCC 10145^T, B. cepacia CIP 80.24^T, B. gladioli pv. gladioli CFBP 2427^T, S. maltophilia CIP 60.77^T, and A. xylosoxidans CIP 61.20. The CE-SSCP patterns of clinical isolates could be strictly superimposed over those of the corresponding reference strains (mobility values of single-strand DNA peaks differed by less than two scan numbers) (data not shown).

Discussion. This is the first study using CE-SSCP analysis for bacterial identification. CE-SSCP analysis technology has several advantages over SSCP analysis with slab gels. Multiple fluorescence labeling simplifies the interpretation of electropherograms. The two strands of DNA in the PCR amplicon were labeled with different fluorescent dyes. Having the two strands labeled with different colors simplifies comparison of data from lane to lane and ensures that the same strands are compared for the various samples. In addition, residual double-stranded products are labeled with both colors and are easily distinguished from single-stranded DNA molecules. Another advantage of the technique is the possibility of controlling lane-to-lane variability by using internal lane size standards labeled with a third fluorescent dye. This makes CE-SSCP analysis highly sensitive for detecting minor DNA changes. CE-SSCP analysis is simple and fast. The only step necessary after PCR amplification is heat denaturation in the presence of formamide. Samples are loaded into 48- or 96-well trays that are processed automatically in the analyzer. Electrophoresis takes only 20 min, giving a throughput of up to 48 samples in a 20-h period. In our laboratory, the ABI PRISM 310 genetic analyzer is utilized for CE-SSCP analysis one night per week. The rest of the time it is used for sequencing. Finally, the cost of CE-SSCP analysis is lower than that for other genotypic methods of identification. Basically, it is a PCR with fluorescently labeled primers. Purification of PCR products on columns, as recommended by the manufacturer, is costly. However, this step can be omitted with no problems provided that the PCR products are sufficiently diluted.