Immunoblots with patient (A) or mice (B) sera against <i>B. petrii</i> LPS.

<p><i>B. petrii</i> LPS was isolated from bacterial pellets by using a LPS extraction kit. <i>B. petrii</i> LPS (10 µg/lane) or commercially obtained <i>E. coli</i> LPS control (3 µg/lane) were electrophoresed on 12% SDS–PAGE, and then transferred to PVDF membrane. The membrane was incubated with the patient serum (1∶5000 dilution) or sera from mice (1∶200 dilution) previously inoculated with <i>B. petrii. B. petrii</i> 1 LPS (4B lanes 2, 3 and 4 for three mice) or <i>B. petrii</i> 3 LPS (4B lanes 6, 7 and 8 for three mice) or with uninoculated normal mouse sera controls (4B lanes 1 and 5). The membrane was then incubated with horseradish peroxidase conjugated sheep anti-human (A) or anti-mice (B) IgG (1∶10000) and blots developed using the enhanced chemiluminescence kit.</p

Immunoblots with patient’s serum against her own and reference strains.

<p>An aliquot containing 10 µg of protein from the soluble (A) and insoluble (B) fractions obtained from <i>B. petrii</i> strains was electrophoresed on 12% SDS–PAGE, and then transferred to PVDF membrane. The membrane was incubated with the patient serum (1∶5000 dilution) obtained ∼2 months after isolation of <i>B. petrii</i> 3 and then horseradish peroxidase conjugated sheep anti-human IgG (1∶10000). The blots were developed using the enhanced chemiluminescence kit. <i>B. petrii</i> 4, <i>B. petrii</i> 4b, <i>B. petrii</i> 4c and <i>B. petrii</i> 4d refer to four different colonies obtained from the primary isolation plate of <i>B. petrii</i> 4. Fig. 2B had a shorter exposure time than Fig. 2A, so bands could be better visualized. If using similar exposure times, the intensity of the bands in Fig. 2B is 48% higher than in Fig. 2A (as determined by densitometric analysis).</p

Molecular typing and growth curves of serially isolated strains of <i>B. petrii.</i>.

<p>(A) The DiversiLab Non fermentor typing kit was used for rep-PCR typing of <i>B. petrii</i> using DNA from clinical and reference strains. Amplicons were detected with the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA) and data analyzed with the DiversiLab software (version 3.3). Results generated include a dendrogram (left) and virtual gel images (right). (B) Genomic DNA was digested with the restriction endonuclease <i>XbaI</i> and separated by PFGE with a CHEF Mapper system. Asterisks indicate band differences among the patient strains. Ladder: Lambda DNA Ladder 48.5 KB–1 MB kb plugs (Lonza). (C) Growth curves were performed on LB broth at 37°C and growth assessed by colony-forming units (CFU) performed with serially diluted aliquots plated on SBA plates. Graph shows mean and SEM from three experiments. <i>B. petrii</i> 1–5: strains of <i>B. petrii</i> serially isolated from our patient; BAA-461: type strain of <i>B. petrii</i> (ATCC BAA-461); 13363: first described clinical strain of <i>B. petrii</i> (NCTC 13363).</p

Serum susceptibility of <i>B. petrii</i> 1 and <i>B. petrii</i> 3.

<p>Bacterial colonies grown on SBA were resuspended in 1% proteose peptone - phosphate-buffered saline (PP-PBS) to a concentration of 1×10⁷ CFU/ml. A 100-µl aliquot was then combined with an equal volume of 10% normal human serum (NHS) diluted in 1% PP-PBS. A 1% PP-PBS (0% serum) solution and heat-inactivated (56°C for 1 hour) normal human serum (HI-NHS) served as controls. Samples were incubated for 2 hours at 37°C with shaking. After incubation, samples were serially diluted, plated onto sheep blood agar plates, and grown 24–48 h at 37°C to determine the number of CFU and calculate % of survival.</p

Adaptability and Persistence of the Emerging Pathogen <i>Bordetella petrii</i>

<div><p>The first described, environmentally isolated, <i>Bordetella petrii</i> was shown to undergo massive genomic rearrangements <i>in vitro</i>. More recently, <i>B. petrii</i> was isolated from clinical samples associated with jaw, ear bone, cystic fibrosis and chronic pulmonary disease. However, the <i>in vivo</i> consequences of <i>B. petrii</i> genome plasticity and its pathogenicity remain obscure. <i>B. petrii</i> was identified from four sequential respiratory samples and a post-mortem spleen sample of a woman presenting with bronchiectasis and cavitary lung disease associated with nontuberculous mycobacterial infection. Strains were compared genetically, phenotypically and by antibody recognition from the patient and from inoculated mice. The successive <i>B. petrii</i> strains exhibited differences in growth, antibiotic susceptibility and recognition by the patient’s antibodies. Antibodies from mice inoculated with these strains recapitulated the specificity and strain dependent response that was seen with the patient’s serum. Finally, we characterize one strain that was poorly recognized by the patient’s antibodies, due to a defect in the lipopolysaccharide O-antigen, and identify a mutation associated with this phenotype. We propose that <i>B. petrii</i> is remarkably adaptable <i>in vivo</i>, providing a possible connection between immune response and bacterial evasion and supporting infection persistence.</p></div

<i>Mycobacterial species</i> isolated, demographic characteristics, symptoms and treatment assigned to each patient.

@<p><i>Mycobacterium species</i> most closely related;</p>*<p>Patients were naïve to TB treatment before they were enrolled in the study and received the TB standard regimen for their disease.</p>$<p>Based on National treatment guidelines for TB, the Standard regimen comprises 2 months of rifampin, isoniazid, pyrazinamide and ethambutol and 4 months of isoniazid and rifampin (2RHZE/4RH). Patients with sputum smears positive at month-5 of standard regimen, receive 1 month of rifampin, isoniazid, pyrazinamide, ethambutol and streptomycin followed by 2 months of rifampin, isoniazid, pyrazinamide, ethambutol and 5 months of rifampin, isoniazid and ethambutol (2RHZES/1RHZE/5RHE called re-treatment regimen). The second line treatment for chronic cases (patients with sputum smears positive after re-treatment regimen) comprises kanamycin, ofloxacin, ethionamide and pyrazinamide (3KOEtZ/18OetZ) for MDR disease. <i>M.af: Mycobacterium africanum</i>; <i>M.tb: Mycobacterium tuberculosis</i>; <i>Neg: negative</i>; <i>Pos: positive</i>; <i>AFB: acid-fast bacilli.</i></p

Impact of NTM on TB sputum smears or cultures.

<p><i>NTM: Nontuberculous mycobacteria</i>; <i>MTBC: Mycobacterium tuberculosis</i> complex;</p>*<p><i>Pos: positive in at least two occasions</i>;</p>#<p><i>Neg: negative in three occasions</i>.</p

Distribution of patients in the naïve and chronic treatment groups based on culture and susceptibility results.

*<p><i>M. tuberculosis</i> complex,</p>#<p>Resistant to isoniazid and rifampin,</p>@<p>Nontuberculous Mycobacteria.</p