Abstract
Accurate identification of gram-negative bacilli from cystic fibrosis (CF) patients is essential. Only 57% (108 of 189) of nonmucoid strains and 40% (24 of 60) of mucoid strains were definitively identified as Pseudomonas aeruginosa with MicroScan Autoscan. Most common misidentifications were Pseudomonas fluorescens-Pseudomonas putida (i.e., the strain was either P. fluorescens or P. putida, but the system did not make the distinction and yielded the result P. fluorescens/putida) and Alcaligenes spp. Extending the incubation to 48 h improved identification, but 15% of isolates remained misidentified. The MicroScan Autoscan system cannot be recommended for the identification of P. aeruginosa isolates from CF patients.
Pseudomonas aeruginosa is the most important cause of lung infections in patients with cystic fibrosis (CF), and over 90% of the mortality in CF is due to chronic infections leading to bronchiectasis and respiratory failure (4). As many as 30% of infants and 80% of adults are infected with P. aeruginosa, but a variety of other nonfermentative multidrug-resistant gram-negative bacilli, such as Burkholderia, Stenotrophomonas, and Achromobacter species, have been recovered from the respiratory tracts of CF patients (1). Accurate identification of isolates recovered from CF patients is essential to guide appropriate antimicrobial therapy and understand the epidemiology of emerging pathogens. Recovery of certain pathogens from patients with CF affects infection control practices (9) and may influence eligibility for lung transplantation (14).
A variety of automated commercial systems for identifying gram-negative bacilli are available. These systems utilize modifications of conventional and chromogenic tests to assign a genus and species designation within 15 to 24 h of incubation. A clinical-site evaluation of the performance of MicroScan panels (Dade International, Inc., West Sacramento, Calif.) demonstrated an overall accuracy of 94% (528 of 562) for P. aeruginosa. However, P. aeruginosa strains, particularly those with the mucoid phenotype, isolated from CF patients often have slower growth rates (5).
This study was designed to examine the ability of the MicroScan Autoscan system to accurately identify strains of P. aeruginosa isolated from CF patients. A total of 249 isolates of P. aeruginosa (189 nonmucoid and 60 mucoid isolates) were examined (3). As previously described, these included CF clinical isolates from Children's Hospital and Regional Medical Center in Seattle, Wash., and multidrug resistant isolates sent to the CF Referral Center for Susceptibility and Synergy Studies at Columbia University in New York, N.Y. (10). Biochemical profiles (i.e., oxidase positive, catalase positive, growth at 42°C, and pigment production) were used to confirm the identification of these strains as P. aeruginosa, and molecular probing with the exotoxin A gene was used for all strains with equivocal results (3).
All procedures using MicroScan Autoscan were performed in accordance with the manufacturer's instructions. In brief, several colonies from sheep blood agar plates were inoculated into brain heart infusion broth and incubated to log phase as recommended for relatively slow-growing bacteria. Suspensions were adjusted to 0.5 McFarland standard and inoculated with a Renok rehydrator into Negative Combo type 15 panels. Plates were read after 20 to 24 h and again at 48 h of incubation on the Autoscan 4 to determine if longer incubation improved the accuracy of identification. Studies of the antimicrobial susceptibility of these isolates have been reported (2).
The initial identification for these 249 nonmucoid and mucoid P. aeruginosa isolates after 20 to 24 h of incubation are shown in Table 1. Only 57% (108 of 189) of nonmucoid strains and 40% (24 of 60) of mucoid strains were definitively identified as P. aeruginosa. An additional 6% (n = 16) of isolates (15 nonmucoid and 1 mucoid) were presumptively identified as P. aeruginosa. As also shown in Table 1, when the incubation was extended to 48 h, identification improved, particularly for mucoid strains. Thus, by 48 h, 86% of nonmucoid and 83% of mucoid strains were correctly identified as P. aeruginosa or presumptive P. aeruginosa. Mucoid strains were more likely to be misidentified than nonmucoid strains (chi square analysis: odds ratio, 1.87 [0.99 to 3.53], P = 0.037).
TABLE 1.
MicroScan identification of Pseudomonas aeruginosa Isolates (N = 249) from CF Patients After 20-24 Hours and 48 Hours of Incubation
| MicroScan identification | No. (%) of isolates at:
|
|||
|---|---|---|---|---|
| 24 h
|
48 h
|
|||
| Nonmucoid (n = 189) | Mucoid (n = 60) | Nonmucoid (n = 66) | Mucoid (n = 35) | |
| P. aeruginosaa | 108 (57) | 24 (40) | 5 (7.5) | 14 (40) |
| Presumptive P. aeruginosaa | 15 (8) | 1 (2) | 34 (51.5) | 11 (31) |
| Total | 123 (65) | 25 (42) | 39 (59) | 25 (71) |
| Pseudomonas spp. | 5 (3) | 5 (8) | 0 | 0 |
| P. fluorescens-P. putida | 12 (6) | 8 (13) | 0 | 0 |
| Burkholderia cepacia | 1 (<1) | 0 | 0 | 0 |
| Stenotrophomonas maltophilia | 1 (<1) | 0 | 1 (1.5) | 0 |
| Alcaligenes spp. | 14 (7) | 4 (7) | 0 | 0 |
| Alcaligenes xylosoxidans | 9 (5) | 3 (5) | 5 (7.5) | 1 (3) |
| Vibrio fluvialis | 0 | 1 (2) | 16 (24) | 7 (20) |
| CDC group IV C-2 | 2 (1) | 0 | 2 (3) | 0 |
| Other | 22 (12)b | 14 (23)c | 3 (5)d | 2 (6)e |
Isolates identified as P. aeruginosa or presumptive P. aeruginosa at 20 to 24 h of incubation were not reread at 48 h.
Includes Vibrio sp. strain SF (n = 5), Acinetobacter lwoffii (n = 4), Ochrobactrum anthropi (n = 2), Moraxella spp. (n = 2), Pasteurella-Actinobacillus spp. (n = 2), Empedobacter brevis (n = 1), Chromobacterium violaceum (n = 1), Enterobacter cloacae (n = 1), Oligella ureolytica (n = 1), Shewanella putrefaciens (n = 1), Shigella spp. (n = 1), and V. parahaemolyticus (n = 1).
Includes Vibrio sp. strain SF (n = 5), Acinetobacter lwoffii (n = 2), Ochrobactrum anthropi (n = 3), Moraxella spp. (n = 1), Pasteurella-Actinobacillus spp. (n = 1), Empedobacter brevis (n = 1), and Pseudomonas stutzeri (n = 1).
Includes one each of Enterobacter cloacae, Shigella spp. and Providencia stuartii.
Includes one each of Yersinia enterocolitica group and Pseudomonas stutzeri.
As demonstrated in Table 2, the most common misidentifications at 24 h were P. fluorescens-P. putida (i.e., the strain was either P. fluorescens or P. putida, but the system did not make the distinction and yielded the result of P. fluorescens/putida (n = 20) and Alcaligenes spp. (n = 30). At 48 h, 12 of 20 (60%) P. fluorescens-P. putida misidentifications and 25 of 30 (83%) Alcaligenes spp. misidentifications were resolved as either P. aeruginosa (n = 8) or presumptive P. aeruginosa (n = 29).
TABLE 2.
Comparison of MicroScan identification at 24 and 48 h of incubation for the most common misidentifications of P. aeruginosa
| 24-h identification | No. of strains
|
48-h identification | No. of strains
|
||
|---|---|---|---|---|---|
| Non-mucoid | Mucoid | Non-mucoid | Mucoid | ||
| P. fluorescens-P. putida | 12 | 8 | P. aeruginosa | 1 | 5 |
| Presumptive P. aeruginosa | 5 | 1 | |||
| Alcaligenes xylosoxidans | 0 | 1 | |||
| Vibrio fluvialis | 6 | 0 | |||
| Yersinia enterocolitica | 0 | 1 | |||
| Alcaligenes spp.a | P. aeruginosa | 1 | 1 | ||
| Presumptive P. aeruginosa | 18 | 5 | |||
| 23 | 7 | Alcaligenes xylosoxidans | 2 | 0 | |
| Vibrio fluvialis | 1 | 1 | |||
| Shigella spp. | 1 | 0 | |||
Of these 30 strains, 9 nonmucoid and 3 mucoid strains were identified as A. xylosoxidans.
These data demonstrated that the MicroScan Autoscan system did not accurately identify CF isolates of P. aeruginosa when used as recommended by the manufacturer; 41% (101 of 249) of strains were misidentified. Prolonging the incubation period to 48 h improved the accuracy of the method, particularly for mucoid strains, but still resulted in 15% (37 of 249) of isolates being misidentified. This relatively poor performance is in accordance with that described for commercial test systems for the identification of other multidrug-resistant bacilli isolated from CF patients (6, 8, 11, 12, 13).
In conclusion, the standard method for the MicroScan Autoscan system cannot be recommended for the identification of CF isolates of nonmucoid P. aeruginosa. It is likely that mucoid strains would be correctly identified by laboratory personnel even when misidentified by the automated system. Prolonging the incubation and/or using biochemical panels or molecular techniques (7, 12) is recommended to improve identification.
Acknowledgments
This study was funded by the U.S. CF Foundation.
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