Bordetella petrii Infection with Long-lasting Persistence in Human

B. petrii infection can persist in persons with chronic obstructive pulmonary disease.

over 1 week to severe coughing with abundant purulent and hemoptoic sputum, although the patient had been treated with respiratory physiotherapy and corticosteroid aerosols at home (which was in poor hygienic condition). C-reactive protein level was only moderately elevated (27 mg/mL, reference <10 mg/mL). This patient had experienced pulmonary tuberculosis 33 years earlier and had been successfully treated. For 30 years, she had diffuse bronchiectasis, which had required a right middle lobectomy 23 years previously. She had a myocardial infarction 6 years ago. The patient also had severe chronic hyponatremia, diagnosed as Schwartz-Bartter syndrome, which may have been related to a chronic respiratory defi ciency. At admission, a purulent sputum sample was taken (class 5 of Bartlett-Murray and Washington criteria) (24) yielded a monomicrobial culture (10 8 CFU/ mL) of B. petrii. After receiving empirical treatment with amoxicillin-clavulanate, the patient improved slowly and was discharged after 2 weeks. Over the next 12 months, the patient returned with bronchorrhea symptoms in November and December 2007 and in May and November 2008. Each time, B. petrii was isolated from purulent sputum specimens in either pure or mixed cultures (in the second sputum culture, B. petrii 10 8 CFU/ mL and Citrobacter freundii 10 4 CFU/mL; in the third sputum culture, B. petrii 10 8 CFU/mL and C. freundii 10 7 CFU/mL; in the fourth culture, B. petrii 10 7 CFU/ mL pure culture; in the fi fth culture, B. petrii 10 7 CFU/ mL and Haemophilus parainfl uenzae 10 7 CFU/mL; and in the last sputum culture, B. petrii 10 7 CFU/mL pure culture). These episodes did not require hospitalization, and the patient's symptoms were empirically treated with amoxicillin and clavulanate. However, during these repetitive episodes, the patient did not have any general symptoms of sepsis (no fever nor elevated C-reactive protein level). The reactivation of tuberculosis and infection with other mycobacteria were excluded by 3 microscopic examinations and liquid and solid cultures. The patient died in January 2009 from deep electrolytic disorders related to Schwartz-Bartter syndrome.

Bacterial Isolation, Identifi cation, DNA Extraction, and Growth Conditions
Sputum samples (25) were plated onto the following agar plates: chocolate PolyViteX agar for bacterial numeration, bromo-cresol purple (BCP) agar plates, selective Haemophilus (chocolate bacitracin) agar, Columbia agar with nalidixic acid and 5% sheep blood (all from bioMérieux, Marcy-l'Etoile, France), and CHROMagar Candida (BBL, Becton, Dickinson and Company, Le Pont de Claix, France). Plates were incubated at 37°C for 24 h and for 48 h in a humidifi ed atmosphere with 9% CO 2 . For each sample, 10 6 -10 8 CFU/mL were observed and characterized. At 48 h, small colonies of a gram-negative bacterium were detected on the fi rst 3 media. Routine identifi cation was performed by a Gram-Negative Identifi cation card on a VITEK 2 automate (bioMérieux) and manually by using API 20NE, API 32GN (bioMérieux), and RapID NH (Remel, Lenexa, KS, USA) strips in accordance with the manufacturer's instructions.
Six isolates were collected sequentially from the patient over 13 months. We chose to analyze the fi rst (October 2007), middle (May 2008), and fi nal (November 2008) isolates from the patient and compared them with the other B. petrii isolates. The Bordetella reference strain and clinical isolates used in this study are listed in Table 1. Bacterial suspensions were prepared from bacteria grown on Bordet-Gengou agar, supplemented with 15% defi brinated blood (BGA, Difco, Detroit, MI, USA) for 24 h at 36°C, which were then resuspended in saline at 1.8 × 10 10 CFU/mL. Sequencing of the 16S rRNA, risA, and ompA Genes DNA from the selected isolates was extracted by using a DNeasy blood and tissue kit (QIAGEN, Courtaboeuf, France). DNA amplifi cation of the 16S rRNA gene was performed as previously described (29,30). Amplifi cation and sequencing of the genes for the Bordetella outer membrane protein A (ompA) and the response regulator (risA) was performed by using the method described by von Wintzingerode et al. (1) with a few modifi cations. To optimize PCR amplifi cation conditions, we designed primers for the ompA gene (ompA3e: 5′-CTC CTC CAA ATT CGC TCT GGC-3′ and ompA4b: 5′-GCA GTT CGC CCT TGC CTT-3′) and risA gene (RisA1c: 5′-AAA ACA CCA ATC CCA TCC GC-3′ and RisA2d: 5′-ACA GGT TGA GCA CAT AGG GC-3′). The nucleotide sequences of 16S rRNA, risA, and ompA genes from the 3 isolates of the patient (FR3799, FR3891, FR3996) and from the other isolate of human origin (FR3497) have been submitted to the EMBL Nucleotide Sequence Database under the respective accession numbers FN691469/FN691470/FN691471/ FN691472; FR669151/FR669152/FR669153/FR669154; and FR669155/FR669156/FR669157/FR669158. For comparative sequence analysis of the 3 different genes, the software, clustalW was used (31).

Matrix-assisted Laser Desorption and Ionization Time-of-Flight (MALDI-TOF) Identifi cation
A MALDI-TOF Axima Assurance (Shimadzu-Biotech Corp., Kyoto, Japan) was used. A positive ion mode, liner mode of detection was running, with a laser frequency of 50 Hz and a mass range window of 2,000-30,000 kDa. The sample was prepared, in duplicate, by a direct deposit of a colony fraction on a target plate, and addition of 1 μL of matrix (acide α-4-cyano-4-hydroxycinnamique, AnagnosTec, Potsdam-Golm, Germany), and drying at ambient temperature. Controls and calibration were done with Escherichia coli CCGU 10979 (Culture Collection Göteborg University, Göteborg, Germany). One hundred spectra were obtained, with Launchpad version 2.8 software for spectrum acquisition (Shimadzu-Biotech Corp). Spectra were analyzed with Saramis software, version 3.3.2 (AnagnosTec).

Additional Tests
Pulsed-fi eld gel electrophoresis (PFGE) analysis was performed as described by Caro et al. (34). Western blot analyses were performed as previously described (35).
Serum specimens used included polyclonal specifi c murine serum specimens (anti-FHA, PRN, PT, AC-Hly) (35), the serum collected 7 months after the hospitalization of the patient infected with B. petrii, and 2 pools of serum samples from patients infected with B. pertussis and B. bronchiseptica, respectively.
Fimbrial protein expression was detected by agglutination with monoclonal anti-Fim2 and anti-Fim-3 antibodies. Cytotoxicity of bacteria to murine alveolar macrophage J774-A1 cells was measured as previously described by Bassinet et al. (36).

Results
The bacteria isolated from the patient grew slowly on the chocolate, Haemophilus chocolate, and BCP agar plates. On routine media, the bacteria were irregular gram negative coccobacillus, nonmotile, and strictly aerobic. They tested positive for oxidase and catalase, were susceptible to colistin, and grew at 36°C, 25°C, and 42°C.
At 48 h to 72 h, cultures on BGA medium showed small, nonhemolytic colonies ≈1 mm in diameter, which were not producing any brown pigment. These tested negative for urease production. The spectra obtained gave a good identifi cation of B. petrii (identifi cation agreement 55.4%).
The sequencing of the 16S rRNA confi rmed the MALDI-TOF identifi cation. The 16S sequences showed 99% of similarity with the 16S rRNA gene sequences from the type strain of environmental origin and the other isolate from human origin. We then performed the sequencing of risA and ompA genes to compare with the genes of the other B. petrii isolates. The risA gene sequences of the clinical isolates showed 93% similarity with the type strain of environmental origin. The species with the next highest similarities were B. bronchiseptica, B. parapapertussis, and B. pertussis, all with a nucleotide identity of 87%. The ompA gene sequences of the clinical isolates demonstrated 89% similarity with the type strain of environmental origin. The species with the next highest similarities were B. bronchiseptica, B. parapapertussis, and B. pertussis, all with a nucleotide identity of 86%. The weaker score obtained with ompA was due to an insertion of 12 nt versus the environmental strain, nucleotides not present in the other isolates from human origin.
The data regarding antimicrobial drug susceptibility obtained are shown in Table 2. Results are compared with other data available from the literature.
As shown in Figure 1, the PFGE patterns obtained with the DNA from the 3 isolates of the reported human casepatient are identical, whereas the patterns obtained with the DNA of the other isolate from human origin and the isolate from the environment show several differences. This indicates that the isolates of the present study are related but part of a different PFGE group.
Results were negative for Fim2 and Fim3 by using the agglutination technique as were results for FHA, PRN, PT, and AC-Hly by Western blot (data not shown) with specifi c antibodies. As shown in Figure 2 In terms of cytoxicity, B. pertussis and B. bronchiseptica are cytotoxic for the J774-A1 macrophages. However, none of the B. petrii isolates were cytotoxic.

Discussion
Identifi cation of B. petrii is still a major problem for clinical laboratories that use automated or manual identifi cation systems. As suggested by Zbinden et al. (37) isolates that do not give a 99% or better typing result should be typed by 16S rRNA sequencing or MALDI-TOF. The spectra obtained here with MALDI-TOF gave an acceptable identifi cation of B. petrii (identifi cation agreement 55.4%). This is a good score, especially because the database contains only 5 spectra of this recently described species because of the low number of isolates available. The phenotypic characteristics of the isolates in our study are similar to those of the few isolates that have been previously described (1,(16)(17)(18)(19).
In a previous study on B. bronchiseptica, we and others working on Bordetella spp. (16) determined that the results obtained for many antimicrobial drugs using the disk diffusion method correlated poorly with clinical therapeutic results and with MICs established using the reference method (32,33; A. Le Coustumier, unpub. data). Fry et al. (16) reported that the clinical isolate was apparently susceptible, by disk diffusion tests, to 5 antimicrobial drugs: clarithromycin, erythromycin, gentamicin, ceftriaxone, and piperacillin+tazobactam. However, the respective reference MICs indicated that only piperacillin+tazobactam was active in vitro with a MIC of 2 μg/mL. Based on the preliminary results the patient received a 6-week course of oral clarithomycin treatment. Despite the successful clinical outcome, the isolate was subsequently shown to be resistant to clarithromycin in vitro. In the only other report (to our knowledge) on a clinical B. petrii isolate, MICs were determined by using VITEK2 Compact (bioMérieux) but MICs of drugs for Bordetella spp. cannot be determined from this database (18). Using Etest strips, a method that has been validated on a wide range of glucose fermenting and nonfermenting gram-negative bacteria, we determined the MIC for 26 widely used antimicrobial drugs from the main therapeutic families (38).
All of the 5 isolates in the present study as well as the isolates described by Fry et al. (16) and Stark et al. (18) appear to have resistance to penicillins (penicillin, amoxicillin), cephalosporins (especially third-generation, extended-spectrum cefotaxime or ceftriaxone and ceftazidime), clindamycin, quinupristin and dalfopristin, rifampin, linezolid, daptomycin, and fucidic acid. We also observed that aminoglycosides had only moderate activity against the bacteria. The isolates in our study also displayed in vitro sensitivity, but low level MICs, to minocyclin, tygecyclin, cotrimoxazole, and fosfomycin.
The large gap in the MICs of amoxicillin and piperacillin between the only environmental isolate available for this study and the clinical isolates may refl ect the inducible response to exposure of the clinical isolates to formerly widely used treatment with β-lactams. Tazobactam does not restore the activity of piperacillin, or even degrade it, probably because of the induction of β-lactamase.
In contrast with the isolate of Fry et al. (16) and the environmental isolate (1), the MICS of carbapenems and systemic fl uoroquinolones were high for the isolates from our patient. No previous treatment with carbapenems could be documented from the long medical history of our patient, although fl uoroquinolones had been frequently prescribed for bronchiectasis. This lack could be partly due to an impermeability-linked cross-resistance between these 2 chemically unrelated families with the common porin mutation, as frequently has been observed for Pseudomonas aeruginosa. 616 Emerging  Using PFGE, we observed that the patterns of the DNA restriction fragments for the different isolates collected from the reported human patient were quite similar. This fi nding confi rms the persistence of the same isolate inside the host. However, several differences are observed with the patterns of the DNA from the environmental or human isolates. These differences could be linked to the loss of pathogenic islands in some of the isolates, as has been recently reported (22).
Using murine serum samples specifi c to the major virulence factors expressed by B. pertussis and pool of sera from patients infected with either B. pertussis, B. bronchiseptica, B. holmesii, or the serum of the current patient infected with B. petrii, we confi rmed that B. petrii isolates do not express FHA, Fim2 and Fim3, PRN, PT, and AC-Hly. The serum sample from the patient infected with B. petrii recognized only 1 protein specifi c to the B. petrii bacterial suspensions derived from the isolates of the clinical patient described in this study. Another protein was specifi c to the 5 B. petrii isolates.
None of the B. petrii isolates were cytotoxic for macrophages. This result was likely because these isolates do not express AC-Hly or BteA.
The source of infection and the pathogenic role of B. petrii are still unknown. For the study patient, the source of infection, just prior to the fi rst episode, was most likely a contamination that occurred during the aerosol therapy performed at home under poor hygienic conditions (according to the patient). This was potentiated by local corticotherapy.
The prevalence of Bordetella spp. within the cystic fi brosis population may well be underestimated, due to the slow growth of this microorganism. However, the prevalence may also be underestimated for all immunosuppressed patients, particularly the elderly. The role that Bordetellae spp. such as bronchiseptica and petrii may play in the progression of pulmonary disease remains unknown, and these species can be misidentifi ed in hospital laboratories (19).