Bordetella holmesii: Comparison of Two Isolates from Blood and a Respiratory Sample

Valérie Bouchez, Nicole Guiso

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Institut Pasteur, Unité Prévention et Thérapie Moléculaires des Maladies Humaines, Paris, France.

DOI: 10.4236/aid.2013.32020                

Abstract

Interest in Bordetella holmesii is increasing, but very little is known about this bacterium, which can be isolated from both blood and respiratory samples. In this study, we compared a B. holmesii isolate from the blood sample of an adult with bacteremia with another isolate from a nasopharyngeal swab from an adult with whooping cough syndrome. Genetic analysis was carried out, targeting relevant genes, and virulence properties were studied in cellular and animal models. Our genomic analysis provided no evidence of traits specific to either blood or respiratory isolates of B. holmesii. Neither isolate was cytotoxic to human tracheal epithelial cells. Both isolates were only weakly invasive and they did not persist within epithelial cells for less than 48 h.

Keywords

Bordetella holmesii; Bacteremia; Respiratory Isolate

1. Introduction

Bordetella holmesii is a Gram-negative bacterium, first described in 1995, following its isolation from the blood of a patient with septicemia [1]. Phylogeny analysis based on 16S rRNA genes initially placed B. holmesii close to B. pertussis. However, multilocus sequence typing showed that B. holmesii did not belong to the classical Bordetella group (B. pertussis, B. parapertussis and B. bronchiseptica), being instead more closely related to B. avium [2] . Very few putative virulence factors have been identified in this species: a two-component bvg operon (bvgA and bvgS) [3,4] , a filamentous hemagglutinin-like protein FHA-bho [5] and an alcaligin operon  ADDIN EN.CITE  ADDIN EN.CITE.DATA [2] . By contrast, no toxin or adhesion similar to those involved in pathogenesis in classical Bordetella species has been identified. There has recently been an increase in the number of cases linking B. holmesii to whooping cough syndromes. The B. holmesii genome harbors insertion sequences, such as IS481, which is also present in the genome of B. pertussis, but at a lower copy number [6]. This poses a potential problem, because the diagnosis of B. pertussis infection is based on the PCR detection of IS481 sequences. The development of specific PCR diagnosis for B. holmesii infection, targeting the recA gene [7-9] , or the transposase IS1001bho [10] , the amplification of specific target as bhoE [11] or the sequencing of 16sRNA or OmpA [12] has made it possible for a number of retrospective studies to demonstrate an increase in the number of reported cases of respiratory infections due to B. holmesii over the last few years [10,11,13-21] . We wondered whether this increase in detection reflected a real increase in the number of infections or was simply a consequence of the increasing use of RT-PCR targeting IS481 for the diagnosis of B. pertussis since 2005. Indeed, Njamkepo et al. [16] reported a prevalence of 6.8% for B. holmesii in respiratory samples from patients with a principal diagnosis of pertussis. Prevalence was highest (20.3%) in adolescents and adults, and no cases were found in children under the age of nine years. By contrast, 1) during an outbreak in Chile, Miranda et al. [22] reported a prevalence of 11.1% for B. holmesii, mostly in adolescents and young adults, but they also identified three cases (3%) in patients under the age of one year; 2) in Argentina in 2010, Bottero et al., [21] reported 9 cases due to B. holmesii, among 1475 pertussis suspected cases patients, 7 under 6 months of age, one 7 months old and one 13 years old. Rodgers et al. [10] recently reported that, during an outbreak of pertussis in Ohio (USA), 30% of the patients were infected with B. holmesii and not with B. pertussis. These findings raise questions concerning the nature of B. holmesii: is it an opportunistic bacterium or an invasive or respiratory pathogen? They also raise questions about the possible differences between invasive and respiratory isolates. The aim of our study was to compare two B. holmesii isolates, one from blood [23] and the other from a respiratory sample [6], in terms of their genetics, targeting relevant genes and considering their virulence in cellular and animal tests.

2. Materials and Methods

2.1. Bacterial Growth and DNA Extraction

All B. pertussis and B. holmesii isolates used were obtained from the Collection of the Pasteur Institute or the French National Reference Center. They were grown at 36˚C for 72 hours on Bordet-Gengou agar (BGA) supplemented with 15% defibrinated sheep blood, and replated on the same medium and incubated for 24 hours before use. A list of the isolates used is presented in Table 1. For pyrosequencing, genomic DNA was prepared on Genomic-tip 500/G anion-exchange columns (Qiagen), according to the manufacturer’s recommendations. For PCR validation, DNA extractions were performed with the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s instructions.

2.2. Pulsed-Field Gel Electrophoresis

DNA fingerprinting was performed by pulsed-field gel electrophoresis (PFGE), as previously described [24].

2.3. Western-Blot Analysis

Western blots were carried out as described by Weber et al. [25].

2.4. Pyrosequencing and In-House Sequence Verifications

Pyrosequencing was carried out with a 454GS-FLX NextGen sequencing platform (Roche Diagnostics GmbH, Beckman Coulter Genomics). Three mate-pair libraries were constructed for both isolates. The two samples were then simultaneously sequenced in one GS-FLX run, with a 70 × 75 mm Pico-Titer plate device (Roche Diagnostics GmbH) and the GS LR-70 sequencing kit (Roche Diagnostics GmbH), as previously described [26] . A standard sequencing assembly was generated with Newbler assembler version 2.3 from 454/Roche, generating 11 scaffolds for Bho1 and 33 for FR4020. However, as no reference annotated genome is available for B. holmesii, we initially focused on the few genes of interest already identified for this species: bvgA/S [3], fhaB  ADDIN EN.CITE  ADDIN EN.CITE.DATA [5] , the alcaligin operon and B. holmesii-specific genes  ADDIN EN.CITE  ADDIN EN.CITE.DATA [2] . Moreover, as pyrosequencing technology is known to generate errors, particularly in homopolymeric nucleotide tracts, we checked all the sequence differences observed in these genes by PCR and classical Sanger sequencing for both the isolates studied and for some additional isolates (Table 1). The primers used are listed in Table 2. Concatenated sequences of the four sequences of interest (located in bvgA/bvgS/fhaB and fimC) were aligned for the construction of a neighbor-joining tree, with PHYLIP software [27] from the Pasteur Mobyle Portal (Figure 1).

We then used the NCBI blastn and blastp programs to identify other genes potentially important for the putative pathogenicity of B. holmesii.

2.5. Cell Culture

The murine monocyte/macrophage-like cell line J774.A1 and human tracheal epithelial cells (HTE) were cultured as previously described  ADDIN EN.CITE  ADDIN EN.CITE.DATA [26]

2.6. Cytotoxicity Assays

Bacterial cytotoxicity to J774.A1 and HTE cells was assessed as previously described  ADDIN EN.CITE  ADDIN EN.CITE.DATA [26] . Briefly, bacteria were added to cells at a ratio of 100 bacteria per cell and gently centrifugated. Infected cells were incubated at 37˚C, in the presence of 5% CO₂, for 8 hours. Cytotoxicity was determined every 2 hours, with the Cytotox 96® Non-Radioactive Cytotoxicity Assay (Promega), which measures the activity of the lactate dehydrogenase released into the culture supernatant. In each experiment with J774.A1, we used Tohama, the B. pertussis reference strain known to be cytotoxic to these cells, as a positive control [28]. In each experiment with HTE cells, we used RB50, the B. bronchiseptica reference strain known to be cytotoxic to these cells, as a positive control [29] .

2.7. HTE Cell Invasion and Persistence Assays

Invasion and persistence assays were conducted with HTE cells, as previously described  ADDIN EN.CITE  ADDIN EN.CITE.DATA [26] . Briefly, bacteria were added to cells at a ratio of 100 bacteria per cell and gently centrifugated. Infected cells were incubated at 37˚C, in the presence of 5% CO₂. After 5 hours of incubation, cells were thoroughly washed and incubated for a further two hours with gentamicin (Sigma) at a final concentration of 100 mg/ml, to kill any remaining extracellular bacteria. For persistence assays, the concentration of gentamicin in the cell culture medium was then decreased to 10 mg/ml over the rest of the incubation period. The number of intracellular Bordetella was determined by cell lysis and determinations of the number of CFU after 5 hours for invasion assays and after 48 hours of incubation for persistence assays.

The results presented are the means and standard deviations of at least three experiments. The symbol (^*) in Figure 2 indicates p < 0.05 in comparisons with the re-

Table 1. List of clinical isolates used in the study.

Table 2. List of primers used in the study.

Four concatenated sequences of interest (located in bvgA/bvgS/fhaB and fimC) were aligned for the construction of a neighbor-joining tree. The symbol (^*) is used to identify isolates of respiratory origin.

Figure 1. Neighbor-joining tree based on partial sequences of bvgA/bvg-S/fhab/fimC.

cently isolated B. pertussis FR3713.

2.8. Animal Models

All procedures involving animals were conducted in accordance with the Institut Pasteur animal care and use committee guidelines. LD₅₀ was determined as previously described  ADDIN EN.CITE  ADDIN EN.CITE.DATA [26] .

3. Results

3.1. Description of the Isolates

Table 1 presents the isolates used in this study. We compared one isolate from a man with sickle cell anemia and B. holmesii bacteremia (Bho1, [23]) with another isolate from the respiratory tract of a woman with whooping cough syndrome (FR4020, [6]). These two isolates had PFGE profiles (data not shown) identical to those of the other isolates tested (Table 1) [6,20,23] . None of the toxins and adhesins involved in the pathogenicity of classical Bordetella species, such as pertussis toxin (PT), adenylate cyclase-hemolysin toxin (ACHly), Bordetella type 3 secretion system effector A (BteA), filamentous hemagglutinin (FHA), pertactin (PRN) and fimbrial proteins (Fim 2 and Fim 3), were detected in suspensions of the isolates with specific antibodies raised against purified B. pertussis virulence factors.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	R. S. Weyant, D. G. Hollis, R. E. Weaver, M. F. M. Amin, A. G. Steigerwalt, S. P. O’Connor, A. M. Whitney, M. I. Daneshvar, C. W. Moss and D. J. Brenner, “Bordetella holmesii sp. nov., a New Gram-Negative Species Associated with Septicemia,” Journal of Clinical Microbiology, Vol. 33, No. 1, 1995, pp. 1-7.
[2]	D. A. Diavatopoulos, C. A. Cummings, H. G. van der Heide, M. van Gent, S. Liew, D. A. Relman and F. R. Mooi, “Characterization of a Highly Conserved Island in the Otherwise Divergent Bordetella holmesii and Bordetella pertussis Genomes,” Journal of bacteriology, Vol. 188, No. 24, 2006, pp. 8385-8394. doi:10.1128/JB.01081-06
[3]	G. Gerlach, S. Janzen, D. Beier and R. Gross, “Functional Characterization of the bvgAS Two-Component System of Bordetella holmesii,” Microbiology, Vol. 150, No. 11, 2004, pp. 3715-3729. doi:10.1099/mic.0.27432-0
[4]	A. Horvat and R. Gross, “Molecular Characterization of the BvgA Response Regulator of Bordetella holmesii,” Microbiology Research, Vol. 164, No. 3, 2009, pp. 243-252. doi:10.1016/j.micres.2006.11.015
[5]	S. Link, K. Schmitt, D. Beier and R. Gross, “Identification and regulation of expression of a gene encoding a filamentous hemagglutinin-related protein in Bordetella holmesii,” BMC Microbiology, Vol. 7, No. 2007, p. 100.
[6]	A. Tizolova, N. Guiso and S. Guillot, “Insertion Sequences Shared by Bordetella Species and Implications for the Biological Diagnosis of Pertussis Syndrome,” European Journal of Clinical Microbiology Infectious Diseases, Vol. 32, No. 1, 2013, pp. 89-96. doi:10.1007/s10096-012-1718-3
[7]	O. Vielemeyer, J. Y. Crouch, S. C. Edberg and J. G. Howe, “Identification of Bordetella pertussis in a Critically Ill Human Immunodeficiency Virus-Infected Patient by Direct Genotypical Analysis of Gram-Stained Material and Discrimination from B. holmesii by Using a Unique recA Gene Restriction Enzyme Site,” Journal of Clinical Microbiology, Vol. 42, No. 2, 2004, pp. 847-849. doi:10.1128/JCM.42.2.847-849.2004
[8]	M. Antila, Q. He, C. de Jong, I. Aarts, H. Verbakel, S. Bruisten, S. Keller, M. Haanpera, J. Makinen, E. Eerola, M. K. Viljanen, J. Mertsola and A. van der Zee, “Bordetella holmesii DNA Is Not Detected in Nasopharyngeal Swabs from Finnish and Dutch Patients with Suspected Pertussis,” Journal of Medical Microbiology, Vol. 55, No. 8, 2006, pp. 1043-1051. doi:10.1099/jmm.0.46331-0
[9]	J. L. Guthrie, A. V. Robertson, P. Tang, F. Jamieson and S. J. Drews, “Novel Duplex Real-Time PCR Assay Detects Bordetella holmesii in Specimens from Patients with Pertussis-Like Symptoms in Ontario, Canada,” Journal of Clinical Microbiology, Vol. 48, No. 4, 2010, pp. 1435-1437. doi:10.1128/JCM.02417-09
[10]	L. Rodgers, S. W. Martin, A. Cohn, J. Budd, M. Marcon, A. Terranella, S. Mandal, D. Salamon, A. Leber, M. L. Tondella, K. Tatti, K. Spicer, A. Emanuel, E. Koch, L. McGlone, L. Pawloski, M. Lemaile-Williams, N. Tucker, R. Iyer, T. A. Clark and M. Diorio, “Epidemiologic and Laboratory Features of a Large Outbreak of Pertussis-Like Illnesses Associated with Co-Circulating Bordetella holmesii and Bordetella pertussis—Ohio, 2010-2011,” Clinical Infectious Diseases, Vol. 56, No. 3, 2013, pp. 322-331. doi:10.1093/cid/cis888
[11]	F. R. Mooi, S. Bruisten, I. Linde, F. Reubsaet, K. Heuvelman, S. van der Lee and J. K. A, “Characterization of Bordetella holmesii Isolates from Patients with Pertussis-Like Illness in the Netherlands,” FEMS Immunology and Medical Microbiology, Vol. 64, No. 2, 2012, pp. 289-291. doi:10.1111/j.1574-695X.2011.00911.x
[12]	S. Jonckheere, T. De Baere, P. Schroeyers, O. Soetens, A. De Bel and I. Surmont, “Prosthetic Valve Endocarditis Caused by Bordetella holmesii, an Acinetobacter Look-Alike,” Journal of Medical Microbiology, Vol. 61, No. 6, 2012, pp. 874-877. doi:10.1099/jmm.0.038695-0
[13]	H. Kamiya, N. Otsuka, Y. Ando, F. Odaira, S. Yoshino, K. Kawano, H. Takahashi, T. Nishida, Y. Hidaka, H. Toyoizumi-Ajisaka, K. Shibayama, K. Kamachi, T. Sunagawa, K. Taniguchi and N. Okabe, “Transmission of Bordetella holmesii during Pertussis Outbreak, Japan,” Emerging Infectious Diseases, Vol. 18, No. 7, 2012, pp. 1166-1169. doi:10.3201/eid1807.120130
[14]	D. M. Livovsky, D. Leibowitz, C. Hidalgo-Grass, V. Temper, S. Salameh and M. Korem, “Bordetella holmesii Meningitis in an Asplenic Patient with Systemic Lupus Erythematosus,” Journal of Medical Microbiology, Vol. 61, No. 8, 2012, pp. 1165-1167. doi:10.1099/jmm.0.038208-0
[15]	E. Mazengia, E. A. Silva, J. A. Peppe, R. Timperi and H. George, “Recovery of Bordetella holmesii from Patients with Pertussis-Like Symptoms: Use of Pulsed-Field Gel Electrophoresis to Characterize Circulating Strains,” Journal of Clinical Microbiology, Vol. 38, No. 6, 2000, pp. 2330-2333.
[16]	E. Njamkepo, S. Bonacorsi, M. Debruyne, S. A. Gibaud, S. Guillot and N. Guiso, “Significant Finding of Bordetella holmesii DNA in Nasopharyngeal Samples from French Patients with Suspected Pertussis,” Journal of Clinical Microbiology, Vol. 49, No. 12, 2011, pp. 4347-4348. doi:10.1128/JCM.01272-11
[17]	W. K. Yih, E. A. Silva, J. Ida, N. Harrington, S. M. Lett and H. George, “Bordetella holmesii-Like Organisms Isolated from Massachussetts Patients with Pertussis-Like Symptoms,” Emerging Infectious Diseases, Vol. 5, No. 3, 1999, pp. 441-443. doi:10.3201/eid0503.990317
[18]	X. Zhang, L. S. Weyrich, J. S. Lavine, A. T. Karanikas and E. T. Harvill, “Lack of Cross-Protection against Bordetella holmesii after Pertussis Vaccination,” Emerging Infectious Diseases, Vol. 18, No. 11, 2012, pp. 1771-1779.
[19]	T. Chambaraud, Z. Dickson, G. Ensergueix, O. Barraud, M. Essig, C. Lacour, J. Allard, F. Bocquentin, J. C. Aldigier and J. P. Rerolle, “Bordetella holmesii Bacteremia in a Renal Transplant Recipient: Emergence of a New Pathogen,” Transplant Infectious Disease, Vol. 14, No. 6, 2012, pp. E134-E136. doi:10.1111/tid.12009
[20]	M. I. Panagopoulos, M. Saint Jean, D. Brun, N. Guiso, S. Bekal, P. Ovetchkine and B. Tapiero, “Bordetella holmesii Bacteremia in Asplenic Children: Report of Four Cases Initially Misidentified as Acinetobacter lwoffii,” Journal of Clinical Microbiology, Vol. 48, No. 10, 2010, pp. 3762-3764. doi:10.1128/JCM.00595-10
[21]	D. Bottero, M. M. Griffith, C. Lara, D. Flores, L. Pianciola, M. E. Gaillard, M. Mazzeo, M. I. Zamboni, M. J. Spoleti, E. Anchart, D. Ruggeri, C. Sorhouet, S. Fiori, M. Galas, M. L. Tondella and D. F. Hozbor, “Bordetella holmesii in Children Suspected of Pertussis in Argentina,” Epidemiology and infection, Vol. 141, No. 4, 2013, pp. 714-717. doi:10.1017/S095026881200132X
[22]	C. Miranda, L. Porte and P. Garcia, “Bordetella holmesii in Nasopharyngeal Samples from Chilean Patients with Suspected Bordetella pertussis Infection,” Journal of Clinical Microbiology, Vol. 50, No. 4, 2012, p. 1505; Author Reply 1506.
[23]	E. Njamkepo, F. Delisle, I. Hagege, G. Gerbaud and N. Guiso, “Bordetella holmesii Isolated from a Patient with Sickle Cell Anemia: Analysis and Comparison with Other Bordetella holmesii Isolates,” Clinical Microbiology and Infection, Vol. 6, No. 3, 2000, pp. 131-136. doi:10.1046/j.1469-0691.2000.00032.x
[24]	V. Caro, E. Njamkepo, S. C. Van Amersfoorth, F. R. Mooi, A. Advani, H. O. Hallander, Q. He, J. Mertsola, M. Riffelmann, C. Vahrenholz, C. H. Von Konig and N. Guiso, “Pulsed-Field Gel Electrophoresis Analysis of Bordetella pertussis Populations in Various European Countries with Different Vaccine Policies,” Microbes Infection, Vol. 7, No. 7-8, 2005, pp. 976-982. doi:10.1016/j.micinf.2005.04.005
[25]	C. Weber, C. Boursaux-Eude, G. Coralie, V. Caro and N. Guiso, “Polymorphism of Bordetella pertussis Isolates Circulating the Last Ten Years in France, Where a Single Effective Whole-Cell Vaccine Has Been Used for More Than Thirty Years,” Journal of Clinical Microbiology, Vol. 39, No. 12, 2001, pp. 4396-4403. doi:10.1128/JCM.39.12.4396-4403.2001
[26]	V. Bouchez, D. Brun, T. Cantinelli, G. Dore, E. Njamkepo and N. Guiso, “First Report and Detailed Characterization of B. pertussis Isolates Not Expressing Pertussis Toxin or Pertactin,” Vaccine, Vol. 27, No. 43, 2009, pp. 6034-6041. doi:10.1016/j.vaccine.2009.07.074
[27]	P. Software, “Phylogeny Inference Package (PHYLIP),” In: J. Felsenstein, Ed., PHYLIP—Phylogeny Inference Package (Version 3.2), Cladistics 5, Department of Genome Sciences, University of Washington, Seattle, 1989, pp. 164-166.
[28]	N. Khelef and N. Guiso, “Induction of Macrophage Apoptosis by Bordetella pertussis Adenylate Cyclase-Hemolysin,” FEMS Microbiology Letters, Vol. 134, No. 1, 1995, pp. 27-32. doi:10.1111/j.1574-6968.1995.tb07909.x
[29]	P. Gueirard, L. Bassinet, I. Bonne, M. C. Prevost and N. Guiso, “Ultrastructural Analysis of the Interactions between Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica and Human Tracheal Epithelial Cells,” Microbial pathogenesis, Vol. 38, No. 1, 2005, pp. 41-46. doi:10.1016/j.micpath.2004.08.003
[30]	S. Mattoo, A. K. Foreman-Wykert, P. A. Cotter and J. F. Miller, “Mechanisms of Bordetella Pathogenesis,” Frontiers in Bioscience, Vol. 6, No. 86, 2001, p. 1.
[31]	M. Sebaihia, A. Preston, D. J. Maskell, H. Kuzmiak, T. D. Connell, N. D. King, P. E. Orndorff, D. M. Miyamoto, N. R. Thomson, D. Harris, A. Goble, A. Lord, L. Murphy, M. A. Quail, S. Rutter, R. Squares, S. Squares, J. Woodward, J. Parkhill and L. M. Temple, “Comparison of the Genome Sequence of the Poultry Pathogen Bordetella avium with Those of B. bronchiseptica, B. pertussis, and B. parapertussis Reveals Extensive Diversity in Surface Structures Associated with Host Interaction,” Journal of Bacteriology, Vol. 188, No. 16, 2006, pp. 6002-6015. doi:10.1128/JB.01927-05
[32]	S. B. Loker, L. M. Temple and A. Preston, “The Bordetella avium BAV1965-1962 Fimbrial Locus Is Regulated by Temperature and Produces Fimbriae Involved in Adherence to Turkey Tracheal Tissue,” Infection and Immunity, Vol. 79, No. 6, 2011, pp. 2423-2429. doi:10.1128/IAI.01169-10
[33]	A. Chahboune, M. Decaffmeyer, R. Brasseur and B. Joris, “Membrane Topology of the Escherichia coli AmpG Permease Required for Recycling of Cell Wall Anhydromuropeptides and AmpC Beta-Lactamase Induction,” Antimicrobial agents and chemotherapy, Vol. 49, No. 3, 2005, pp. 1145-1149. doi:10.1128/AAC.49.3.1145-1149.2005
[34]	W. E. Goldman and B. T. Cookson, “Structure and Functions of the Bordetella Tracheal Cytotoxin,” The Tokai Journal of Experimental and Clinical Medicine, Vol. 13, 1988, pp. 187-191.
[35]	K. M. Yen, M. R. Karl, L. M. Blatt, M. J. Simon, R. B. Winter, P. R. Fausset, H. S. Lu, A. A. Harcourt and K. K. Chen, “Cloning and Characterization of a Pseudomonas mendocina KR1 Gene Cluster Encoding Toluene-4-Monooxygenase,” Journal of Bacteriology, Vol. 173, No. 17, 1991, pp. 5315-5327.
[36]	R. Gross, C. A. Guzman, M. Sebaihia, V. A. dos Santos, D. H. Pieper, R. Koebnik, M. Lechner, D. Bartels, J. Buhrmester, J. V. Choudhuri, T. Ebensen, L. Gaigalat, S. Herrmann, A. N. Khachane, C. Larisch, S. Link, B. Linke, F. Meyer, S. Mormann, D. Nakunst, C. Ruckert, S. Schneiker-Bekel, K. Schulze, F. J. Vorholter, T. Yevsa, J. T. Engle, W. E. Goldman, A. Puhler, U. B. Gobel, A. Goesmann, H. Blocker, O. Kaiser and R. Martinez-Arias, “The Missing Link: Bordetella petrii Is Endowed with Both the Metabolic Versatility of Environmental Bacteria and Virulence Traits of Pathogenic Bordetellae,” BMC Genomics, Vol. 9, 2008, p. 449.
[37]	M. Lechner, K. Schmitt, S. Bauer, D. Hot, C. Hubans, E. Levillain, C. Locht, Y. Lemoine and R. Gross, “Genomic Island Excisions in Bordetella petrii,” BMC Microbiology, Vol. 9, 2009, p. 141.
[38]	K. E. Stockbauer, A. K. Foreman-Wykert and J. F. Miller, “Bordetella Type III Secretion Induces Caspase 1-Independent Necrosis,” Cell Microbiology, Vol. 5, No. 2, 2003, pp. 123-132. doi:10.1046/j.1462-5822.2003.00260.x
[39]	L. Bassinet, P. Gueirard, B. Maitre, B. Housset, P. Gounon and N. Guiso, “Role of Adhesins and Toxins in Invasion of Human Tracheal Epithelial Cells by Bordetella pertussis,” Infection and Immunity, Vol. 68, No. 4, 2000, pp. 1934-1941. doi:10.1128/IAI.68.4.1934-1941.2000
[40]	Y. Ishibashi, D. A. Relman and A. Nishikawa, “Invasion of Human Respiratory Epithelial Cells by Bordetella pertussis: Possible Role for a Filamentous Hemagglutinin Arg-Gly-Asp Sequence and Alpha5beta1 Integrin,” Microbial Pathogenesis, Vol. 30, No. 5, 2001, pp. 279-288. doi:10.1006/mpat.2001.0432
[41]	T. Spilker, A. A. Liwienski and J. J. LiPuma, “Identification of Bordetella spp. in Respiratory Specimens from Individuals with Cystic Fibrosis,” Clinical Microbiology Infection, Vol. 14, No. 5, 2008, pp. 504-506. doi:10.1111/j.1469-0691.2008.01968.x
[42]	A. T. Harrington, J. A. Castellanos, T. M. Ziedalski, J. E. Clarridge 3rd and B. T. Cookson, “Isolation of Bordetella avium and Novel Bordetella Strain from Patients with Respiratory Disease,” Emerging Infectious Diseases, Vol. 15, No. 1, 2009, pp. 72-74. doi:10.3201/eid1501.0716