Superiority of molecular techniques for identification of gram-negative, oxidase-positive rods, including morphologically nontypical Pseudomonas aeruginosa, from patients with cystic fibrosis

Phenotypic identification of gram-negative bacteria from Cystic Fibrosis (CF) patients carries a high risk of misidentification. Therefore, we compared the results of biochemical identification by API 20NE with 16S rRNA gene sequencing in 88 gram-negative, oxidase-positive rods, other than morphologically and biochemically typical P. aeruginosa, from respiratory secretions of CF patients. The API 20NE allowed correct identification of the bacterial species in 15 out of 88 (17%) isolates investigated. Agreement between the API and the 16S rRNA gene sequencing results was high only in isolates with an API result classified as "excellent identification". Even API results classified as "very good identification" or "good identification" showed a high rate of misidentification (67% and 84%). Fifty-two isolates of morphological and biochemical nontypical Pseudomonas aeruginosa, representing 59% of all isolates investigated, were not identifiable or misidentified in the API 20NE. Therefore, rapid molecular diagnostic techniques like real-time PCR and fluorescence in situ hybridization (FISH) were evaluated in this particular group of bacteria for identification of the clinically most relevant pathogen, P. aeruginosa. The LightCycler PCR assay with a P. aeruginosa-specific probe showed a sensitivity and specificity of 98.1% and 100%, respectively. For FISH analysis, a newly designed P. aeruginosa-specific probe had a sensitivity and specificity of 100%. In conclusion, molecular methods are superior over biochemical tests for identification of gram-negative, oxidase-positive rods in CF patients. In addition, real-time PCR and FISH allowed identification of morphologically nontypical isolates of P. aeruginosa within a few hours.     

Identification and Antimicrobial Susceptibility of Alcaligenes xylosoxidans Isolated from Patients with Cystic Fibrosis

In the past decade, potential pathogens, including Alcaligenes species, have been increasingly recovered from cystic fibrosis (CF) patients. Accurate identification of multiply antibiotic-resistant gram-negative bacilli is critical to understanding the epidemiology and clinical implications of emerging pathogens in CF. We examined the frequency of correct identification of Alcaligenes spp. by microbiology laboratories affiliated with American CF patient care centers. Selective media, an exotoxin A probe for Pseudomonas aeruginosa, and a commercial identification assay, API 20 NE, were used for identification. The activity of antimicrobial agents against these clinical isolates was determined. A total of 106 strains from 78 patients from 49 CF centers in 22 states were studied. Most (89%) were correctly identified by the referring laboratories as Alcaligenes xylosoxidans. However, 12 (11%) strains were misidentified; these were found to be P. aeruginosa (n = 10), Stenotrophomonas maltophilia (n = 1), and Burkholderia cepacia (n = 1). Minocycline, imipenem, meropenem, piperacillin, and piperacillin-tazobactam were the most active since 51, 59, 51, 50, and 55% of strains, respectively, were inhibited. High concentrations of colistin (100 and 200 microg/ml) inhibited 92% of strains. Chloramphenicol paired with minocycline and ciprofloxacin paired with either imipenem or meropenem were the most active combinations and inhibited 40 and 32%, respectively, of strains. Selective media and biochemical identification proved to be useful strategies for distinguishing A. xylosoxidans from other CF pathogens. Standards for processing CF specimens should be developed, and the optimal method for antimicrobial susceptibility testing of A. xylosoxidans should be determined.     

Capillary Electrophoresis–Single-Strand Conformation Polymorphism Analysis for Rapid Identification of Pseudomonas aeruginosa and Other Gram-Negative Nonfermenting Bacilli Recovered from Patients with Cystic Fibrosis

We used capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) analysis of PCR-amplified 16S rRNA gene fragments for rapid identification of Pseudomonas aeruginosa and other gram-negative nonfermenting bacilli isolated from patients with cystic fibrosis (CF). Target sequences were amplified by using forward and reverse primers labeled with various fluorescent dyes. The labeled PCR products were denatured by heating and separated by capillary gel electrophoresis with an automated DNA sequencer. Data were analyzed with GeneScan 672 software. This program made it possible to control lane-to-lane variability by standardizing the peak positions relative to internal DNA size markers. Thirty-four reference strains belonging to the genera Pseudomonas, Brevundimonas, Burkholderia, Comamonas, Ralstonia, Stenotrophomonas, and Alcaligenes were tested with primer sets spanning 16S rRNA gene regions with various degrees of polymorphism. The best results were obtained with the primer set P11P-P13P, which spans a moderately polymorphic region (Escherichia coli 16S rRNA positions 1173 to 1389 [M. N. Widjojoatmodjo, A. C. Fluit, and J. Verhoef, J. Clin. Microbiol. 32:3002-3007, 1994]). This primer set differentiated the main CF pathogens from closely related species but did not distinguish P. aeruginosa from Pseudomonas alcaligenes-Pseudomonas pseudoalcaligenes and Alcaligenes xylosoxidans from Alcaligenes denitrificans. Two hundred seven CF clinical isolates (153 of P. aeruginosa, 26 of Stenotrophomonas maltophilia, 15 of Burkholderia spp., and 13 of A. xylosoxidans) were tested with P11P-P13P. The CE-SSCP patterns obtained were identical to those for the corresponding reference strains. Fluorescence-based CE-SSCP analysis is simple to use, gives highly reproducible results, and makes it possible to analyze a large number of strains. This approach is suited for the rapid identification of the main gram-negative nonfermenting bacilli encountered in CF.     

Restriction enzyme and Southern hybridization analyses of Pseudomonas aeruginosa strains from patients with cystic fibrosis.

Conventional typing schemes for Pseudomonas aeruginosa may not discriminate strains. P. aeruginosa isolates from patients with cystic fibrosis were examined by restriction enzyme analysis and Southern hybridization. There was marked diversity of restriction enzyme analysis patterns among P. aeruginosa DNAs from cystic fibrosis isolates; however, sequential isolates obtained from individual patients showed very little variation over an 8-year period. DNA fragments from the alginate biosynthesis gene complex, the exotoxin A gene, and the elastase gene of P. aeruginosa were used in Southern hybridization analysis. The patterns of hybridization to the elastase and algD gene probes were highly conserved in all isolates, therefore, these DNA fragments are not useful in discriminating strains, in contrast to the exotoxin A gene probe.     

PCR-based assay for differentiation of Pseudomonas aeruginosa from other Pseudomonas species recovered from cystic fibrosis patients

Pseudomonas aeruginosa is the major opportunistic bacterial pathogen in persons with cystic fibrosis (CF); pulmonary infection occurs in approximately 80% of adult CF patients. Much of CF patient management depends on accurate identification of P. aeruginosa from sputum culture. However, identification of this species may be problematic due to the marked phenotypic variability demonstrated by CF sputum isolates and the presence of other closely related species. To facilitate species identification, we used 16S ribosomal DNA (rDNA) sequence data to design PCR assays intended to provide genus- or species-level identification. Both assays yielded DNA fragments of the predicted size. We tested 42 culture collection strains (including 14 P. aeruginosa strains and 28 strains representing 16 other closely related Pseudomonas species) and 43 strains that had been previously identified as belonging to 28 nonpseudomonal species also recovered from CF patient sputum. Based on these 85 strains, the specificity and sensitivity of both assays were 100%. To further assess the utility of the PCR assays, we tested 66 recent CF sputum isolates. The results indicated that preliminary phenotypic testing had misidentified several isolates. The 16S rDNA sequence was determined for 38 isolates, and in all cases it confirmed the results of the PCR assays. Thus, we have designed two PCR assays: one is specific for the genus Pseudomonas, while the other is specific for P. aeruginosa. Both assays show 100% sensitivity and specificity.     

Evaluation of MicroScan Autoscan for identification of Pseudomonas aeruginosa isolates from cystic fibrosis patients

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.     

PCR identification of Pseudomonas aeruginosa and direct detection in clinical samples from cystic fibrosis patients.

This report describes a PCR primer pair that targets the algD GDP mannose gene of Pseudomonas aeruginosa and produces a specific 520-bp PCR product useful for P. aeruginosa identification. This PCR assay was tested with 182 isolates of P. aeruginosa and 20 isolates of other bacterial species, and demonstrated 100% specificity and sensitivity. The test was also able to detect P. aeruginosa directly in clinical samples such as sputum or throat swabs obtained from cystic fibrosis patients. The combination of this primer with a universal bacterial primer, acting as a control to assess DNA quality in the sample, resulted in a robust PCR method that can be used for rapid P. aeruginosa detection.     

Direct detection and identification of Pseudomonas aeruginosa in clinical samples such as skin biopsy specimens and expectorations by multiplex PCR based on two outer membrane lipoprotein genes, oprI and oprL.

A multiplex PCR test based on the simultaneous amplification of two lipoprotein genes, oprI and oprL, was designed and evaluated for its ability to directly detect fluorescent pseudomonads (amplification of oprI open reading frame, 249 bp) and Pseudomonas aeruginosa (amplification of oprL open reading frame, 504 bp) in clinical material. A collection of reference strains including 20 different species of fluorescent pseudomonads was tested. Positive PCR results for both genes were observed only for P. aeruginosa isolates (n = 150), including strains of clinical and environmental origin, while only one gene, oprI, was amplified from the other fluorescent pseudomonads. All other bacteria tested (n = 15) were negative by the amplification test. The lower detection level for P. aeruginosa was estimated to be 10(2) cells/ml. Preliminary evaluation on testing skin biopsy specimens from patients with burns (n = 14) and sputum samples from cystic fibrosis patients (n = 49) and other patients (n = 19) showed 100% sensitivity and 74% specificity in comparison with culture. This multiplex PCR assay appears promising for the rapid and sensitive detection of P. aeruginosa in clinical specimens. Further evaluation of its specificity in longitudinal clinical studies is warranted.     

Evaluation of an immunofluorescent-antibody test for rapid identification of Pseudomonas aeruginosa in blood cultures.

An immunofluorescent-antibody test was developed for rapid detection of Pseudomonas aeruginosa in blood cultures. The test uses a murine monoclonal antibody specific for all strains of P. aeruginosa. In initial tests, bright uniform immunofluorescence signals were seen when each of the 17 international serotypes, as well as 14 additional isolates of P. aeruginosa, were examined. No immunofluorescent staining was observed when 37 other gram-negative and 15 gram-positive species were studied. In a clinical study, the assay was applied to broth smears of 86 gram-negative bacilli isolated from 74 bacteremic patients and 28 additional clinical isolates of Pseudomonas sp. and other oxidase-positive gram-negative bacilli recovered from various body sites. Smears were made directly from blood cultures which were positive for gram-negative bacilli by Gram staining. Eleven (15%) of 74 patients with gram-negative bacteremia had a positive test for P. aeruginosa. Including the results of these 11 isolates recovered in a prospective study and an additional 10 isolates from a retrospective study, we obtained a sensitivity and specificity of 100% (21 positive specimens and 103 negative specimens, respectively). These preliminary results suggest that this is a useful reagent for rapid presumptive identification of P. aeruginosa in blood cultures. With the immunofluorescent-antibody test, P. aeruginosa could be identified within 1 h of Gram stain evidence of gram-negative bacteremia.     

Evaluation of the Oxi/Ferm tube system for identification of nonfermentative Gram-negative bacilli.

The Oxi/Ferm tube system designed for identification of oxidative-fermentative gram-negative bacteria was evaluated for identification of nonfermentative gram-negative bacilli by comparing it with the conventional system. The nine biochemical reactions used as the initial tests in the Oxi/Ferm tube system showed a close agreement with the same conventional reactions. However, the system was only 41% accurate to species or biotype for complete identification of 239 isolates which were well distributed in 48 species and biotypes and included organisms not listed in the Oxi/Ferm tube identification list. The system correctly identified 56% of the test organisms when the degree of identification was based on the manufacturer's guidance list. However, all isolates of Acinetobacter calcoaceticus, Bordetella bronchiseptica, Pseudomonas aeruginosa, P. diminuta, and group IIf were completely identified by the system.     

Clinical evaluation of a direct fluorescent monoclonal antibody test for detection of Pseudomonas aeruginosa in blood cultures.

A direct fluorescent monoclonal antibody test (DFA; Genetic Systems Corp., Seattle, Wash.) was evaluated for the detection of Pseudomonas aeruginosa in 178 blood culture broths obtained from 128 patients. The DFA identified 44 (98%) of 45 blood cultures positive for P. aeruginosa and was negative in 131 (98%) of 133 blood cultures which grew gram-negative rods other than P. aeruginosa. Upon further investigation, saline suspensions of the organism from the false-negative blood culture were strongly (4+) DFA positive. The false-positive reactions were not due to cross-reactivity, as shown by lack of DFA staining of the non-P. aeruginosa isolates following subculture to agar media. The specificity of the reagent was further demonstrated by directly staining culture isolates including 10 serotypes of P. aeruginosa (all positive) and 57 selected gram-negative bacilli including eight species of Pseudomonas that were not P. aeruginosa (all negative). DFA staining of blood culture broths was easy to perform and read with minimal background fluorescence. The DFA method can be performed in 50 min and appears promising as a rapid method for the identification of P. aeruginosa bacteremia.     

Characterisation of aerobic gram-negative bacteria from subgingival sites of dogs--potential bite wound pathogens

Ninety-eight aerobic, gram-negative bacterial isolates from subgingival samples from family-owned dogs with naturally occurring periodontitis were characterised phenotypically by conventional biochemical testing, by cellular fatty acid profiling and by the use of commercial identification systems. The majority (48, 81%) of the fermentative isolates but only 18% of the non-fermenters were identified by conventional biochemical testing alone. With additional cellular fatty acid profiling, another 7 (12%) fermentative and 23 (59%) non-fermentative isolates were identified to genus or group level. Cellular fatty acid analysis was essential for the identification of most non-fermenters, many of which are difficult to identify due to a paucity of positive reactions in routine biochemical tests. Commercial identification systems were less useful and did not contribute to further identification of these problematic isolates. This study underlines the difficulties encountered in the identification of canine oral bacteria--a group of potential bite wound pathogens--and presents schemes for microbiology laboratories to characterise such isolates.     

Accuracy of four commercial systems for identification of Burkholderia cepacia and other gram-negative nonfermenting bacilli recovered from patients with cystic fibrosis.

Burkholderia cepacia has recently been recognized as an important pathogen in chronic lung disease in patients with cystic fibrosis (CF). Because of the social, psychological, and medical implications of the isolation of B. cepacia from CF patients, accurate identification of this organism is essential. We compared the accuracies of four commercial systems developed for the identification of nonfermenting, gram-negative bacilli with that of conventional biochemical testing for 150 nonfermenters including 58 isolates of B. cepacia recovered from respiratory secretions from CF patients. The accuracies of the four systems for identifying all nonfermenters ranged from 57 to 80%, with the RapID NF Plus system being most accurate. The accuracies of these systems for identifying B. cepacia ranged from 43 to 86%, with the Remel system being most accurate. Depending on the commercial system, from two to seven isolates were misidentified as B. cepacia. The relatively poor performance of the commercial systems requires that identification of certain nonfermenters be confirmed by conventional biochemical testing. These organisms include B. cepacia, Burkholderia sp. other than B. cepacia, and infrequently encountered environmental species (Pseudomonas and Flavobacterium species). In addition, conventional biochemical testing should be done if a commercial system fails to assign an identification to an organism. Confirmatory testing should preferably be performed by a reference laboratory with experience in working organisms isolated from CF patients.     

Epidemiology of chronic Pseudomonas aeruginosa infections in cystic fibrosis.

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.     

Comparison of Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometer to BD Phoenix automated microbiology system for identification of gram-negative bacilli

We compared the BD Phoenix automated microbiology system to the Bruker Biotyper (version 2.0) matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) system for identification of gram-negative bacilli, using biochemical testing and/or genetic sequencing to resolve discordant results. The BD Phoenix correctly identified 363 (83%) and 330 (75%) isolates to the genus and species level, respectively. The Bruker Biotyper correctly identified 408 (93%) and 360 (82%) isolates to the genus and species level, respectively. The 440 isolates were grouped into common (308) and infrequent (132) isolates in the clinical laboratory. For the 308 common isolates, the BD Phoenix and Bruker Biotyper correctly identified 294 (95%) and 296 (96%) of the isolates to the genus level, respectively. For species identification, the BD Phoenix and Bruker Biotyper correctly identified 93% of the common isolates (285 and 286, respectively). In contrast, for the 132 infrequent isolates, the Bruker Biotyper correctly identified 112 (85%) and 74 (56%) isolates to the genus and species level, respectively, compared to the BD Phoenix, which identified only 69 (52%) and 45 (34%) isolates to the genus and species level, respectively. Statistically, the Bruker Biotyper overall outperformed the BD Phoenix for identification of gram-negative bacilli to the genus (P < 0.0001) and species (P = 0.0005) level in this sample set. When isolates were categorized as common or infrequent isolates, there was statistically no difference between the instruments for identification of common gram-negative bacilli (P > 0.05). However, the Bruker Biotyper outperformed the BD Phoenix for identification of infrequently isolated gram-negative bacilli (P < 0.0001).     

Use of a genus- and species-specific multiplex PCR for identification of enterococci

The 16S rRNA gene has previously been used to develop genus-specific PCR primers for identification of enterococci. In addition, the superoxide dismutase gene (sodA) has been identified as a potential target for species differentiation of enterococci. In this study, Enterococcus genus-specific primers developed by Deasy et al. (E1/E2) were incorporated with species-specific primers based upon the superoxide dismutase (sodA) gene for development of a multiplex PCR. This assay provides simultaneous genus and species identification of 23 species of enterococci using seven different reaction mixtures. Accuracy of identification of the multiplex PCR was determined by comparisons to standard biochemical testing, the BBL Crystal kit, VITEK, and API Rapid ID 32 Strep. Isolates from swine feces, poultry carcasses, environmental sources, and retail food were evaluated and, overall, results for 90% of the isolates tested by PCR agreed with results obtained using standard biochemical testing and VITEK. Eighty-five percent and 82% of PCR results agreed with results from the API Rapid ID 32 Strep and BBL Crystal tests, respectively. With the exception of concurrence between identification using standard biochemical testing and VITEK (85%) and between BBL Crystal and VITEK (83%), the percent agreement for PCR was higher than or equal to any other pairwise comparison. Multiplex PCR for genus and species determination of enterococci provides an improved, rapid method for identification of this group of bacteria.     

Identification of Pseudomonas aeruginosa by pyocyanin production on Tech agar.

Pseudomonas aeruginosa is the only gram-negative bacillus capable of producing the very distinctive water-soluble pigment pyocyanin. We evaluated the reliability of this characteristic as a unique test for the identification of this organism by using Tech agar (BBL Microbiology Systems, Cockeysville, Md.) medium. A retrospective and prospective analysis was performed with a total of 835 strains of P. aeruginosa; 818 (98%) produced pigment within 48 h of incubation, and 96% of those which produced pigment were positive after overnight incubation. Seventeen strains (2.0%) failed to produce pigment; 15 were mucoid strains from patients with cystic fibrosis. Tech agar is an effective, simple, and inexpensive medium for P. aeruginosa identification and may be used as a unique test for all potential P. aeruginosa isolates (beta hemolytic on blood agar; lactose-negative, oxidase-positive colonies). Nonpigmented mucoid strains, as well as other nonpigmented organisms, will require additional testing to ensure proper identification.     

Rapid detection and identification of metallo-beta-lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis

Metallo-beta-lactamase enzymes (MbetaL) are encoded by transferable genes, which appear to spread rapidly among gram-negative bacteria. The objective of this study was to develop a multiplex real-time PCR assay followed by a melt curve step for rapid detection and identification of genes encoding MbetaL-type enzymes based on the amplicon melting peak. The reference sequences of all genes encoding IMP and VIM types, SPM-1, GIM-1, and SIM-1 were downloaded from GenBank, and primers were designed to obtain amplicons showing different sizes and melting peak temperatures (Tm). The real-time PCR assay was able to detect all MbetaL-harboring clinical isolates, and the Tm-assigned genotypes were 100% coincident with previous sequencing results. This assay could be suitable for identification of MbetaL-producing gram-negative bacteria by molecular diagnostic laboratories.     

Chromosomal mechanisms of aminoglycoside resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients

In total, 40 Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients were included in this study. Twenty of these were collected in 1994 and 1997, from six CF patients, and the rest were collected from different CF patients in 2000 and 2001. The relative expression of mRNA for the efflux pump protein MexY was determined by real-time PCR and correlated with susceptibilities to amikacin and tobramycin. The chromosomal genes mexZ , rplY , galU , PA5471 and nuoG , which were found to have a role in the gradual increase in MICs of aminoglycoside antibiotics in laboratory mutants of P. aeruginosa , were analysed. MexY mRNA overproduction was found in 17/20 isolates collected in 1994 and 1997, and was correlated with decreased susceptibility to aminoglycosides. Alteration of the MexXY–OprM efflux system has been the main mechanism of resistance to aminoglycoside antibiotics in CF P. aeruginosa isolates over the 3-year period. In several isolates, expression of the PA5471 gene product might have some effect on elevated MICs of aminoglycosides. Inactivation of rplY , galU and/or nuoG may explain the gradual increase in MICs of aminoglycosides in laboratory mutants but probably not in the CF environment, as rplY and galU were unaltered in all isolates, and nuoG was not expressed in only one isolate. No 16S rRNA A-site mutations were found in any of the four copies of the gene in 13 investigated isolates.     

Early immune response to the components of the type III system of Pseudomonas aeruginosa in children with cystic fibrosis

The lungs of patients with cystic fibrosis (CF) are colonized initially by Pseudomonas aeruginosa, which is associated with progressive lung destruction and increased mortality. The pathogenicity of P. aeruginosa is caused by a number of virulence factors, including exotoxin A (ETA) and the type III cytotoxins (ExoS, ExoT, ExoU, and ExoY). P. aeruginosa contacts the plasma membrane to deliver type III cytotoxins through a channel formed by PopB, PopD, and PcrV; ETA enters mammalian cells via receptor-mediated endocytosis. The Wisconsin CF Neonatal Screening Project is a longitudinal investigation to assess the potential benefits and risks of newborn screening for CF; the project was the source of serum samples used in this study. Past studies evaluated the longitudinal appearance of antibodies to ETA and elastase and P. aeruginosa infections in patients with CF. The current study characterized the longitudinal appearance of antibodies to components of the type III system in children with CF. Western blot analyses showed that serum antibodies to PopB, PcrV, and ExoS were common. Longitudinal enzyme-linked immunosorbent assays determined that the first detection of antibodies to pooled ExoS/PopB occurred at a time similar to those of detection of antibodies to a P. aeruginosa cell lysate and the identification of oropharyngeal cultures positive for P. aeruginosa. This indicates that children with CF are colonized early with P. aeruginosa expressing the type III system, implicating it in early pathogenesis, and implies that surveillance of clinical symptoms, oropharyngeal cultures, and seroconversion to type III antigens may facilitate early detection of P. aeruginosa infections.