From August 2005 through June 2008, sputum samples, bronchoalveolar lavage samples, or both, were collected from patients with CF at 2 cystic fibrosis treatment centers (CFTCs), Timone Children’s Hospital (patients <18 years of age; CFTC1) and Ste. Marguerite Hospital in Marseille (patients >18 years of age; CFTC2). Only samples that showed, by direct Gram staining, infrequent epithelial cells (<10 cells/field) and numerous polymorphonuclear cells (>25 cells/field) were further analyzed and processed according to current local guidelines (4). A portion of each sample was also frozen at –20°C for further study. Respiratory samples from patients who did not have CF (children admitted to the pediatric healthcare center and adults admitted to CFTC2) were also collected for control analysis. The Corynebacterium reference strains used in this study are listed in the Table. This study was approved by our local ethics committee (no. 07–011).

The positive bacilli from respiratory samples, identified by Gram stain, were investigated by metabolic tests, as oxidase and catalase activities, and by the use of Api (RAPID) Coryne Database 2.0 system (bioMérieux, Marcy-l’Etoile, France) (15). The antimicrobial drug susceptibility testing was performed by disk diffusion method on Muelle-Hinton agar with 5% sheep blood incubated for 24 h at 37°C.

Primers used in this study for amplification and sequencing the partial rpoB gene as well as PCR methods have been previously described (16). Multiple sequence alignment and percentage of similarities for the partial rpoB genes between the different species of corynebacteria were done by using the ClustalW program on the EMBL-EBI web server (www.ebi.ac.uk/clustalw). A phylogenic tree was generated by using the neighbor-joining method from MEGA 4.0 software (www.megasoftware.net). Kimura 2-parameter was used as a substitution model to construct the rpoB tree. Bootstrap replicates were performed to estimate the reliabilities of the nodes of the phylogenic tree obtained.

The strains were plated on Columbia agar with 5% sheep blood (COS) (bioMérieux) and incubated for 24 h at 37°C. One isolated colony from each strain was harvested and deposited on a target plate (Bruker Daltonics, Bremen, Germany) in 3 replicates. Two microliters of matrix solution (saturated α-cyano-4-hydroxycinnamic acid, 50% acetonitrile, 2.5% trifluoroacetic acid) was then added and samples were processed in the MALDI-TOF mass spectrometry (337 nm) (Autoflex, Bruker Daltonics with the flex control software) (10). The profiles were compared and analyzed by Biotyper 2.0 (Bruker Daltonics) and finally a dendrogram of mass spectral data was constructed by using the instructor default setting. The Biotyper 2.0 program generates the tree depending on distance-based method and does not provide branch support values.

A new real-time PCR with a TaqMan probe (Applied Biosystems) that targets the rpoB gene of C. pseudodiphtheriticum has been developed and tested retrospectively in sputum samples that had previously been collected in a 1-year study from January through December 2006. Sputum samples from 4 groups of patients were analyzed: sputum samples from child (group 1) and adult (group 2) CF patients and sputum samples from non-CF children (group 3) and from non-CF adults (group 4). Primers and probe used were as follows: CorynPF (5′-GACGGYGCTTCCAACGAAGA-3′) and CorynPR (5′-CCGACGGAGATCGGGTGC-3′) and probe CorynPr (6FAM-TCTGTTGGCTAACTCCCGYCCAAA-TAMRA). Specificity of these primers and probe was verified in silico by using the BLAST program (www.ncbi.nlm.nih.gov/BLAST) as well as by using corynebacteria reference strains (Table). Sensitivity was assessed by using tenfold serial dilutions of a 0.5 MacFarland inoculum.

Overall, 229 patients with CF were monitored from August 2005 through June 2008 in the 2 CFTCs in Marseille (118 children and 111 adults). During this period, 18 corynebacteria were isolated from respiratory samples of 13 children with CF (11.0%) but none from adults with CF (p<0.001). Details for the 13 patients are given in the Table A1. The mean age was 4.3 (0.3–16) years, and the sex ratio (M:F) was 0.6. Isolation of C. pseudodiphtheriticum was associated with clinical symptoms in 10 patients (76.9%), including cough, rhinitis, asthma crisis, and lung exacerbations (Table A1). The culture of C. pseudodiphtheriticum from respiratory samples was pure in 6 cases (in 2 cases, patients had clinical symptoms). For 4 patients, a Corynebacterium isolate was obtained on >1 occasion (Table A1). Six patients were treated, including 3 with β-lactams only, 1 with a combination of a β-lactam and cotrimoxazole, 1 with cotrimoxazole alone, and 1 with cotrimoxazole alone initially and then amoxicillin because no improvement was noticed and the isolate was resistant to cotrimoxazole.

All corynebacteria were isolated from Columbia agar with 5% sheep blood. Colonies were white and nonhemolytic. They were all catalase positive and oxidase negative. The use of the ApiCoryne 2.0 system yielded identification of 16 C. pseudodiphtheriticum and 1 C. propinquum, with a confidence level 83%–99% (Table A1). The remaining isolate was poorly identified as Brevibacter sp. with an uninterpretable pattern (Table A1). All isolates were susceptible to β-lactams, vancomycin, rifampin, gentamicin, and doxycycline, whereas there was heterogeneity of susceptibility for erythromycin and cotrimoxazole (Table A1). The partial rpoB gene sequencing provided an accurate identification for 18 isolates (A1 to M) with similarity >97% compared with reference strains (Table A1). The results of the MALDI-TOF identification matched perfectly with the partial rpoB sequencing identification for all the isolates; mean score values ranged from 1.8–2.5 (Table A1). Figure 1 presents 2 trees built by using MALDI-TOF mass spectrometry (Figure 1, panel A) and by using partial rpoB gene sequences (Figure 1, panel B). Although comparison between these 2 trees was impossible because of the different methods used (i.e., Euclidean distance method for MALDI-TOF dendrogram and neighbor-joining method for phylogenetic tree), the 2 trees gave a similar clustering of the isolates (Figure 1).

Sensitivity of our new real-time PCR ranged from 5 CFU/mL to 10 CFU/mL; specificity for C. pseudodiphtheriticum was verified by testing Corynebacterium spp. reference strain cultures with C. propinquum, the most closely related species, which was also amplified. A low level of cross-amplification was also observed in 3 other species with cycle thresholds (Ct) >38 cycles, including C. accolens, C. durum, and C. riegelii (Table); this finding could not be considered clinically relevant. To estimate the prevalence of this bacterium in our CF population, we used the real-time PCR to test, retrospectively and blindly, all respiratory samples available from January through December 2006 from 146 patients with CF (n = 356 sputum samples; 86 children, group 1; and 60 adults, group 2) and from 56 patients without CF (n = 67 sputum samples; 18 children, group 3; and 38 adults, group 4). We found 24 PCR-positive sputum specimens (Ct <37) in 17 children (19.8%) and 3 adults (5%) in patients with CF (Figure 2). Among these 24 PCR-positive samples only 2 were culture positive (sample A1, Table A1) (p<0.0001). Thus, 16 additional children and 3 adult patients with CF were eventually colonized with this bacterium. For the control group, although all samples were culture negative, we found 3 PCR-positive samples in 2 adult patients, who were followed up in CFTC2 for lung transplantation, and none from children (Figure 2). The 2 PCR-positive lung-transplant patients were hospitalized in the adult CFTC with 2 adult patients with CF during the same period. For these 2 patients, cultures of sputum samples were polymicrobial, and findings were interpreted as normal flora. Finally, the number of PCR-positive children with CF was significantly higher than the number of either children without CF or adults with CF (p = 0.03 and p = 0.01, respectively; Figure 2). Conversely, this difference was not significant between adult patients with CF and adult patients without CF (Figure 2). Notably, the 18 children who did not have CF were seen by clinicians in the same hospital, but not in the same healthcare center, and were not in contact with CF children. Thus, the only risk factor found for being infected or colonized with C. pseudodiphtheriticum was to be monitored at the CF center.