Use of 16S rRNA gene sequencing for identification of nonfermenting gram-negative bacilli recovered from patients attending a single cystic fibrosis center

During 1999, we used partial 16S rRNA gene sequencing for the prospective identification of atypical nonfermenting gram-negative bacilli isolated from patients attending our cystic fibrosis center. Of 1,093 isolates of nonfermenting gram-negative bacilli recovered from 148 patients, 46 (4.2%) gave problematic results with conventional phenotypic tests. These 46 isolates were genotypically identified as Pseudomonas aeruginosa (19 isolates, 12 patients), Achromobacter xylosoxidans (10 isolates, 8 patients), Stenotrophomonas maltophilia (9 isolates, 9 patients), Burkholderia cepacia genomovar I/III (3 isolates, 3 patients), Burkholderia vietnamiensis (1 isolate), Burkholderia gladioli (1 isolate), and Ralstonia mannitolilytica (3 isolates, 2 patients), a recently recognized species.     

Use of 16S rRNA gene sequencing for identification of "Pseudomonas-like" isolates from sputum of patients with cystic fibrosis

Since nonfermenting, Gram negative bacilli recovered from patients with cystic fibrosis could be misidentified with phenotypic procedures, we used partial 16S ribosomal RNA gene (16S gene) sequencing to identify these "Pseudomonas-like" isolates. 473 isolates were recovered from 66 patients in 2003. Sequencing was used to identify 29 (from 24 patients) of the 473 isolates, showing unclear results with routine tests. PCR with specific primers was carried out to amplify a 995 bp fragment, which was then sequenced. The sequences were analyzed with GenBank database for species assignment. Phenotypic and genotypic results were concordant for 20/29 isolates (10 Pseudomonas aeruginosa, 5 Burkholderia cepacia, 3 Stenotrophomonas maltophilia, 2 Achromobacter xylosoxidans). However, 3 of the 5 B. cepacia isolates were then identified as Burkholderia multivorans with a PCR-RFLP procedure. Phenotypic misidentification was observed for 9/29 isolates: 4 A. xylosoxidans, 1 P. aeruginosa, 1 Bordetella petrii, 1 Bordetella bronchiseptica, 1 Ralstonia respiraculi and 1 Ralstonia mannitolilytica. Partial 16S gene sequencing improved the identification of "Pseudomonas-like" isolates from cystic fibrosis patients, but the accuracy to distinguish between genomovars of the B. cepacia complex was inadequate.     

Identification of Pandoraea Species by 16S Ribosomal DNA-Based PCR Assays

The recently described genus Pandoraea contains five named species (Pandoraea apista, Pandoraea pulmonicola, Pandoraea pnomenusa, Pandoraea sputorum, and Pandoraea norimbergensis) and four unnamed genomospecies. Pandoraea spp. have mainly been recovered from the respiratory tracts of cystic fibrosis (CF) patients. Accurate genus- and species-level identification by routine clinical microbiology methods is difficult, and differentiation from Burkholderia cepacia complex organisms may be especially problematic. This can have important consequences for the management of CF patients. On the basis of 16S ribosomal DNA sequences, PCR assays for the identification of Pandoraea spp. were developed. A first PCR assay was developed for the identification of Pandoraea isolates to the genus level. PCR assays for the identification of P. apista and P. pulmonicola as a group, P. pnomenusa, P. sputorum, and P. norimbergensis were also developed. All five assays were evaluated with a panel of 123 bacterial isolates that included 69 Pandoraea sp. strains, 24 B. cepacia complex strains, 6 Burkholderia gladioli strains, 9 Ralstonia sp. strains, 5 Alcaligenes xylosoxidans strains, 5 Stenotrophomonas maltophilia strains, and 5 Pseudomonas aeruginosa strains. The use of these PCR assays facilitates the identification of Pandoraea spp. and avoids the misidentification of a Pandoraea sp. as a B. cepacia complex isolate.     

In vitro antimicrobial activity of "last-resort" antibiotics against unusual nonfermenting Gram-negative bacilli clinical isolates

In this prospective multicentric study, we assessed the in vitro antimicrobial activity of carbapenems (imipenem, meropenem, and doripenem), tigecycline, and colistin against 166 unusual nonfermenting Gram-negative bacilli (NF-GNB) clinical isolates collected from nine French hospitals during a 6-month period (from December 1, 2008, to May 31, 2009). All NF-GNB isolates were included, except those phenotypically identified as Pseudomonas aeruginosa or Acinetobacter baumannii. Minimal inhibitory concentrations (MICs) of antimicrobial agents were determined by using the E-test technique. The following microorganisms were identified: Stenotrophomonas maltophilia (n=72), Pseudomonas spp. (n=30), Achromobacter xylosoxidans (n=25), Acinetobacter spp. (n=18), Burkholderia cepacia complex (n=9), Alcaligenes faecalis (n=7), and Delftia spp. (n=5). All isolates of Acinetobacter spp., A. faecalis, and Delftia spp. were susceptible to the three carbapenems. Imipenem exhibited the lowest MICs against Pseudomonas spp., and meropenem, as compared with imipenem and doripenem, displayed an interesting antimicrobial activity against A. xylosoxidans and B. cepacia complex isolates. Conversely, no carbapenem exhibited any activity against S. maltophilia. Except for S. maltophilia isolates, tigecycline and colistin exhibited higher MICs than carbapenems, but covered most of the microorganisms tested in this study. To our knowledge, no prior study has compared antimicrobial activity of these five antibiotics, often considered as "last-resort" treatment options for resistant Gram-negative infections, against unusual NF-GNB clinical isolates. Further studies should be carried out to assess the potential clinical use of these antibiotics for the treatment of infections due to these microorganisms.     

Evaluation of the colorimetric VITEK 2 card for identification of gram-negative nonfermentative rods: comparison to 16S rRNA gene sequencing

Ninety strains of a collection of well-identified clinical isolates of gram-negative nonfermentative rods collected over a period of 5 years were evaluated using the new colorimetric VITEK 2 card. The VITEK 2 colorimetric system identified 53 (59%) of the isolates to the species level and 9 (10%) to the genus level; 28 (31%) isolates were misidentified. An algorithm combining the colorimetric VITEK 2 card and 16S rRNA gene sequencing for adequate identification of gram-negative nonfermentative rods was developed. According to this algorithm, any identification by the colorimetric VITEK 2 card other than Achromobacter xylosoxidans, Acinetobacter sp., Burkholderia cepacia complex, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia should be subjected to 16S rRNA gene sequencing when accurate identification of nonfermentative rods is of concern.     

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.     

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.     

Fourier transform infrared spectroscopy for rapid identification of nonfermenting gram-negative bacteria isolated from sputum samples from cystic fibrosis patients

The accurate and rapid identification of bacteria isolated from the respiratory tract of patients with cystic fibrosis (CF) is critical in epidemiological studies, during intrahospital outbreaks, for patient treatment, and for determination of therapeutic options. While the most common organisms isolated from sputum samples are Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, in recent decades an increasing fraction of CF patients has been colonized by other nonfermenting (NF) gram-negative rods, such as Burkholderia cepacia complex (BCC) bacteria, Stenotrophomonas maltophilia, Ralstonia pickettii, Acinetobacter spp., and Achromobacter spp. In the present study, we developed a novel strategy for the rapid identification of NF rods based on Fourier transform infrared spectroscopy (FTIR) in combination with artificial neural networks (ANNs). A total of 15 reference strains and 169 clinical isolates of NF gram-negative bacteria recovered from sputum samples from 150 CF patients were used in this study. The clinical isolates were identified according to the guidelines for clinical microbiology practices for respiratory tract specimens from CF patients; and particularly, BCC bacteria were further identified by recA-based PCR followed by restriction fragment length polymorphism analysis with HaeIII, and their identities were confirmed by recA species-specific PCR. In addition, some strains belonging to genera different from BCC were identified by 16S rRNA gene sequencing. A standardized experimental protocol was established, and an FTIR spectral database containing more than 2,000 infrared spectra was created. The ANN identification system consisted of two hierarchical levels. The top-level network allowed the identification of P. aeruginosa, S. maltophilia, Achromobacter xylosoxidans, Acinetobacter spp., R. pickettii, and BCC bacteria with an identification success rate of 98.1%. The second-level network was developed to differentiate the four most clinically relevant species of BCC, B. cepacia, B. multivorans, B. cenocepacia, and B. stabilis (genomovars I to IV, respectively), with a correct identification rate of 93.8%. Our results demonstrate the high degree of reliability and strong potential of ANN-based FTIR spectrum analysis for the rapid identification of NF rods suitable for use in routine clinical microbiology laboratories.     

Ribosomal DNA-directed PCR for identification of Achromobacter (Alcaligenes) xylosoxidans recovered from sputum samples from cystic fibrosis patients

The opportunistic human pathogen Achromobacter (Alcaligenes) xylosoxidans has been recovered with increasing frequency from respiratory tract culture of persons with cystic fibrosis (CF). However, confusion of this species with other closely related respiratory pathogens has limited studies to better elucidate its epidemiology, natural history, and pathogenic role in CF. Misidentification of A. xylosoxidans as Burkholderia cepacia complex is especially problematic and presents a challenge to effective infection control in CF. To address the problem of accurate identification of A. xylosoxidans, we developed a PCR assay based on a 16S ribosomal DNA sequence. In an analysis of 149 isolates that included 47 A. xylosoxidans and several related glucose-nonfermenting species recovered from CF sputum, the sensitivity and specificity of this PCR assay were determined to be 100 and 97%, respectively. The availability of this assay will enhance identification of A. xylosoxidans, thereby facilitating study of the pathogenic role of this species and improving infection control efforts in CF.     

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.     

Development of a PCR probe test for identifying Pseudomonas aeruginosa and Pseudomonas (Burkholderia) cepacia.

AIMS--To develop a system of species specific polymerase chain reaction (PCR) and DNA hybridisation based on 16s ribosomal RNA sequences for the identification of Pseudomonas aeruginosa and Pseudomonas (Burkholderia) cepacia in sputum from children with cystic fibrosis. METHODS--Most of the 16s rRNA sequences from strains of Ps aeruginosa, Ps (Burkholderia) cepacia, and Ps putida were determined. PCR primers and DNA probes were synthesised from suitable sequences and then evaluated on bacterial cultures and sputum samples. RESULTS--About 1000 bases of sequence was obtained from strains of Ps aeruginosa, Ps (Burkholderia) cepacia, and Ps putida. PCR of bacterial cultures was species specific, but PCR on sputum resulted in some non-specific amplification products. The subsequent hybridisation reaction was species specific. CONCLUSION--A species specific system of PCR and DNA hybridisation based on 16s rRNA sequences is applicable in clinical practice, and may aid the early diagnosis of respiratory tract infection with small numbers of Ps aeruginosa and Ps (Burkholderia) cepacia in patients with cystic fibrosis.     

16S rRNA gene sequencing versus the API 20 NE system and the VITEK 2 ID-GNB card for identification of nonfermenting Gram-negative bacteria in the clinical laboratory

Over a period of 26 months, we have evaluated in a prospective fashion the use of 16S rRNA gene sequencing as a means of identifying clinically relevant isolates of nonfermenting gram-negative bacilli (non-Pseudomonas aeruginosa) in the microbiology laboratory. The study was designed to compare phenotypic with molecular identification. Results of molecular analyses were compared with two commercially available identification systems (API 20 NE, VITEK 2 fluorescent card; bioMérieux, Marcy l'Etoile, France). By 16S rRNA gene sequence analyses, 92% of the isolates were assigned to species level and 8% to genus level. Using API 20 NE, 54% of the isolates were assigned to species and 7% to genus level, and 39% of the isolates could not be discriminated at any taxonomic level. The respective numbers for VITEK 2 were 53%, 1%, and 46%, respectively. Fifteen percent and 43% of the isolates corresponded to species not included in the API 20 NE and VITEK 2 databases, respectively. We conclude that 16S rRNA gene sequencing is an effective means for the identification of clinically relevant nonfermenting gram-negative bacilli. Based on our experience, we propose an algorithm for proper identification of nonfermenting gram-negative bacilli in the diagnostic laboratory.     

Development of rRNA-Based PCR Assays for Identification of Burkholderia cepacia Complex Isolates Recovered from Cystic Fibrosis Patients

PCR assays targeting rRNA genes were developed to identify species (genomovars) within the Burkholderia cepacia complex. Each assay was tested with 177 bacterial isolates that also underwent taxonomic analysis by whole-cell protein profile. These isolates were from clinical and environmental sources and included 107 B. cepacia complex strains, 23 Burkholderia gladioli strains, 20 Ralstonia pickettii strains, 10 Pseudomonas aeruginosa strains, 8 Stenotrophomonas maltophilia strains, and 9 isolates belonging to nine other species. The sensitivity and specificity of the 16S rRNA-based assay for Burkholderia multivorans (genomovar II) were 100 and 99%, respectively; for Burkholderia vietnamiensis (genomovar V), sensitivity and specificity were 87 and 92%, respectively. An assay based on 16S and 23S rRNA gene analysis of B. cepacia ATCC 25416 (genomovar I) was useful in identifying genomovars I, III, and IV as a group (sensitivity, 100%, and specificity, 99%). Another assay, designed to be specific at the genus level, identified all but one of the Burkholderia and Ralstonia isolates tested (sensitivity, 99%, and specificity, 96%). The combined use of these assays offers a significant improvement over previously published PCR assays for B. cepacia.     

Species-specific PCR as a tool for the identification of Burkholderia gladioli

Burkholderia gladioli colonizes the respiratory tracts of patients with cystic fibrosis and chronic granulomatous disease. However, due to the high degree of phenotypic similarity between this species and closely related species in the Burkholderia cepacia complex, accurate identification is difficult. Incorrect identification of these species may have serious repercussions for the management of patients with cystic fibrosis. To develop an accurate procedure for the identification of B. gladioli, a molecular method to discriminate between this species and other species commonly isolated from the sputa of patients with cystic fibrosis was investigated. The 23S ribosomal DNA was cloned from several clinical isolates of B. gladioli, and the nucleotide sequence was determined. Computer-assisted sequence comparisons indicated four regions of the 23S rRNA specific for this species; these regions were used to design three primer pairs for species-specific PCR. Two of the primer pairs showed 100% sensitivity and specificity for B. gladioli when tested against a panel of 47 isolates comprising 19 B. gladioli isolates and 28 isolates of 16 other bacterial species. One of the primer pairs was further assessed for species specificity by using a panel of 102 isolates obtained from the Burkholderia cepacia Research Laboratory and Repository. The species-specific PCR was positive for 70 of 74 isolates of B. gladioli and was negative for all other bacterial species examined. Overall, this primer pair displayed a sensitivity and specificity of 96% (89 of 93) and 100%, respectively. These data demonstrate the potential of species-specific PCR for the identification of B. gladioli.     

Identification of Burkholderia spp. in the clinical microbiology laboratory: comparison of conventional and molecular methods.

Cystic fibrosis (CF) predisposes patients to bacterial colonization and infection of the lower airways. Several species belonging to the genus Burkholderia are potential CF-related pathogens, but microbiological identification may be complicated. This situation is not in the least due to the poorly defined taxonomic status of these bacteria, and further validation of the available diagnostic assays is required. A total of 114 geographically diverse bacterial isolates, previously identified in reference laboratories as Burkholderia cepacia (n = 51), B. gladioli (n = 14), Ralstonia pickettii (n = 6), B. multivorans (n = 2), Stenotrophomonas maltophilia (n = 3), and Pseudomonas aeruginosa (n = 11), were collected from environmental, clinical, and reference sources. In addition, 27 clinical isolates putatively identified as Burkholderia spp. were recovered from the sputum of Dutch CF patients. All isolates were used to evaluate the accuracy of two selective growth media, four systems for biochemical identification (API 20NE, Vitek GNI, Vitek NFC, and MicroScan), and three different PCR-based assays. The PCR assays amplify different parts of the ribosomal DNA operon, either alone or in combination with cleavage by various restriction enzymes (PCR-restriction fragment length polymorphism [RFLP] analysis). The best system for the biochemical identification of B. cepacia appeared to be the API 20NE test. None of the biochemical assays successfully grouped the B. gladioli strains. The PCR-RFLP method appeared to be the optimal method for accurate nucleic acid-mediated identification of the different Burkholderia spp. With this method, B. gladioli was also reliably classified in a separate group. For the laboratory diagnosis of B. cepacia, we recommend parallel cultures on blood agar medium and selective agar plates. Further identification of colonies with a Burkholderia phenotype should be performed with the API 20NE test. For final confirmation of species identities, PCR amplification of the small-subunit rRNA gene followed by RFLP analysis with various enzymes is recommended.     

Arbitrarily primed PCR and sequencing of 16S rDNA for epidemiological typing and species identification of Burkholderia cepacia isolates from Swedish patients with cystic fibrosis reveal genetic heterogeneity

To investigate whether arbitrarily primed (AP)-PCR and/or 16S rDNA sequencing could be used as rapid methods for epidemiological typing and species identification of clinical Burkholderia isolates from patients with cystic fibrosis (CF), a total of 39 clinical B. cepacia isolates, including 33 isolates from 14 CF patients, were fingerprinted. ERIC-2 primer was used for AP-PCR. The AP-PCR clustering analysis resulted in 14 different clusters at a 70% similarity level. The AP-PRC patterns were individual despite considerable similarities. To sequence rDNA, a broad-range PCR was applied. The PCR product included four variable loops (V8, V3, V4 and V9) of the 16S ribosomal small subunit RNA gene. The multiple sequence alignment produced 12 different patterns, 5 of them including more than one isolate. Heterogeneity of the bases in the V3 region, indicating the simultaneous presence of at least two different types of 16S rRNA genes in the same cell, was revealed in 10 isolates. Most of the CF patients were adults who had advanced disease at follow-up. Both the sequencing and the AP-PCR patterns revealed genetic heterogeneity of isolates between patients. According to the results obtained, AP-PCR could advantageously be used for epidemiological typing of Burkholderia, whereas partial species identification could effectively be obtained by sequencing of the V3 region of the 16S RNA gene.     

Identification and detection of Stenotrophomonas maltophilia by rRNA-directed PCR

Stenotrophomonas maltophilia has recently emerged as an important nosocomial pathogen in immunocompromised patients, in transplant recipients, and in persons with cystic fibrosis (CF). While this organism is nonpathogenic in healthy individuals, it is increasingly associated with morbidity and mortality in susceptible populations. Recent studies have indicated that for approximately 10% of CF patients with moderate lung disease, S. maltophilia can be cultured from respiratory tract secretions. Identification of S. maltophilia can be problematic, and analysis of isolates from the Burkholderia cepacia Research Laboratory and Repository showed that several isolates presumptively identified as B. cepacia by clinical microbiology laboratories were in fact S. maltophilia. To overcome the problems associated with definitive identification, we developed species-specific PCR (SS-PCR) primers, designated SM1 and SM4, directed to the 23S rRNA gene, and tested their utility to accurately identify S. maltophilia directly from sputum. The SS-PCR was developed and tested against a panel of 112 S. maltophilia isolates collected from diverse geographic locations. To test for specificity, 43 isolates from 17 different species were analyzed. PCR with the SM1-SM4 primer pair and isolated genomic DNA as a template resulted in amplification of a band from all S. maltophilia isolates and was uniformly negative for all other species tested, yielding a sensitivity and a specificity of 100% for the SS-PCR. The utility of the SS-PCR to directly identify S. maltophilia in sputum was examined. Thirteen expectorated sputum samples from CF patients were analyzed by SS-PCR. Three samples were PCR positive, in complete concordance with the conventional laboratory culture. Thus, we have developed an SS-PCR protocol that can rapidly and accurately identify S. maltophilia isolates and which can be used for the direct detection of this organism in CF patient sputum.     

Identification of Burkholderia cepacia isolates from patients with cystic fibrosis and use of a simple new selective medium.

We evaluated 819 isolates referred to us as "Burkholderia cepacia" from cystic fibrosis (CF) clinics and research laboratories from five countries; 28 (3.4%) were not B. cepacia. A further 12 (1.5%) organisms appeared to be other Burkholderia species, but identification could not be confirmed by conventional means. The most prevalently misidentified organisms were Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, and Comamonas acidovorans. Many of these organisms grew on oxidation-fermentation polymyxin-bacitracin-lactose (OFPBL) and Pseudomonas cepacia agars, selective media currently used for B. cepacia isolation. We developed a new medium, B. cepacia selective agar (BCSA), which is more enriched for the growth of B. cepacia yet which is more selective against other organisms than currently available selective agars. A total of 190 of 191 (99.5%) isolates of B. cepacia from patients with CF grew on BCSA without vancomycin, whereas 100% grew on OFPBL agar and 179 (94.2%) grew on P. cepacia agar. Of 189 other gram-negative and gram-positive organisms tested, 10 (5.3%) grew on BCSA without vancomycin. The addition of vancomycin to BCSA lowered the false positivity rate to 3.7% without further inhibition of B. cepacia. The false positivity rates for OFPBL and P. cepacia agars were 19.6 and 13.8%, respectively. Isolates of B. cepacia from CF patients grew most quickly on BCSA, with 201 of 205 (98.0%) being readily visible within 24 h, whereas 182 (88.8%) grew on OFPBL agar and 162 (79.0%) grew on P. cepacia agar within 24 h. We propose that the use of BCSA will allow investigators to overcome many of the difficulties associated with the identification of B. cepacia and should be considered for use as a primary isolation agar for specimens from patients with CF.     

Lipopolysaccharide (LPS) from Burkholderia cepacia is more active than LPS from Pseudomonas aeruginosa and Stenotrophomonas maltophilia in stimulating tumor necrosis factor alpha from human monocytes

Whole cells and lipopolysaccharides (LPSs) extracted from Burkholderia cepacia, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Escherichia coli were compared in their ability to stimulate tumor necrosis factor alpha (TNF-alpha) from the human monocyte cell line MonoMac-6. B. cepacia LPS, on a weight-for-weight basis, was found to have TNF-alpha-inducing activity similar to that of LPS from E. coli, which was approximately four- and eightfold greater than the activity of LPSs from P. aeruginosa and S. maltophilia, respectively. The LPS-stimulated TNF-alpha production from monocytes was found to be CD14 dependent. These results suggest that B. cepacia LPS might play a role in the pathogenesis of inflammatory lung disease in cystic fibrosis, and in some patients it might be responsible, at least in part, for the sepsis-like cepacia syndrome.     

Home-use nebulizers: a potential primary source of Burkholderia cepacia and other colistin-resistant, gram-negative bacteria in patients with cystic fibrosis.

Inhalation of aerosols contaminated with gram-negative bacteria generated from home-use nebulizers used by cystic fibrosis (CF) patients may be a primary route for bacterial colonization of the lung. Burkholderia cepacia was isolated from 3 of [corrected] 35 home-use nebulizers, and Stenotrophomonas maltophilia was isolated from 4 of 35 home-use nebulizers. Sputum cultures for two patients whose nebulizers were contaminated with B. cepacia did not yield the organism. However, DNA macrorestriction analysis by pulsed-field gel electrophoresis confirmed that one of two strains of B. cepacia recovered from the nebulizer of a third patient was also present in the sputum of that patient. Although Pseudomonas aeruginosa was isolated from 34 patients, none of the nebulizers were positive for the organism. Sixty-nine percent of nebulizers were contaminated, and up to 16 different environmental colistin-resistant, gram-negative species were identified. The heaviest contamination was found beneath the chamber atomizer. A questionnaire survey showed that the majority of patients (28 of 34) were receiving nebulized colistin and/or gentamicin. Patients who followed recommended instructions for good nebulizer hygienic practice and paid particular attention to drying had minimal or no contamination of their nebulizers.