Bordetella bronchiseptica is a potent and safe adjuvant that enhances the antigen-presenting capability of dendritic cells

We previously demonstrated that Bordetella bronchiseptica (B. bronchiseptica) antigen (Ag) enhances the Mycoplasma hyopneumoniae Ag-specific immune response. The focus of this study was whether acellular bacterin of B. bronchiseptica could be used as an adjuvant to increase antigen-presenting capability of dendritic cells (DCs) by increasing the level of activation. The metabolic activity of DCs was increased by B. bronchiseptica, similar to lipopolysaccharide (LPS). Flow cytometry analysis revealed that B. bronchiseptica increases the expression of major histocompatibility complex class-2, cluster of differentiation (CD)40, CD54, and CD86 which are closely related to DC-mediated immune responses. B. bronchiseptica enhanced the production of cytokines related to adaptive immune responses. Furthermore, the survival rate of B. bronchiseptica-injected groups was 100% at 15 and 20 mg/kg doses, whereas that of LPS-injected groups was only 20%, 0% at 15 and 20 mg/kg doses respectively, and so B. bronchiseptica is likely to be safer than LPS. Taken together, these results indicate that B. bronchiseptica can be used as an adjuvant to enhance the antigen-presenting capability of DCs. B. bronchiseptica is a candidate for producing vaccines, especially in case of DC-mediating efficacy and safety demands. This study provides researchers and clinicians with valuable information regarding the usage of B. bronchiseptica as a safe bacteria-derived immunostimulating agent for developing efficient vaccines.

Antimicrobial resistance in bacteria isolated from pigs with respiratory clinical signs in Brazil / Resistência a antimicrobianos em bactérias isoladas de suínos com sinais clínicos respiratórios no Brasil

Antimicrobial resistance is a current and important issue to public health, and it is usually associated with the indiscriminate use of antimicrobials in animal production. This study aimed to evaluate the antimicrobial susceptibility profile in bacterial isolates from pigs with clinical respiratory signs in Brazil. One hundred sixty bacterial strains isolated from pigs from 51 pig farms in Brazil were studied. In vitro disk-diffusion method was employed using 14 antimicrobial agents: amoxicillin, penicillin, ceftiofur, ciprofloxacin, enrofloxacin, chlortetracycline, doxycycline, oxytetracycline, tetracycline, erythromycin, tilmicosin, florfenicol, lincomycin, and sulfadiazine/trimethoprim. The majority of isolates were resistant to at least one antimicrobial agent (98.75%; 158/160), while 31.25% (50/160) of the strains were multidrug resistant. Streptococcus suis and Bordetella bronchiseptica were the pathogens that showed higher resistance levels. Haemophilus parasuis showed high resistance levels to sulfadiazine/trimethoprim (9/18=50%). We observed that isolates from the midwestern and southern regions exhibited four times greater chance of being multidrug resistant than the isolates from the southeastern region studied. Overall, the results of the present study showed a great level of resistance to lincomycin, erythromycin, sulfadiazine/trimethoprim, and tetracycline among bacterial respiratory pathogens isolated from pigs in Brazil. The high levels of antimicrobial resistance in swine respiratory bacterial pathogens highlight the need for the proper use of antimicrobials in Brazilian pig farms.(AU)

A resistência antimicrobiana é uma questão atual e muito importante para a saúde pública, geralmente associada ao uso indiscriminado de antimicrobianos na produção animal. Diante disso, foi investigado o perfil de sensibilidade-antimicrobiana em isolados bacterianos de suínos com sinais clínicos respiratórios no Brasil. Foram estudadas 96 isolados provenientes de 51 granjas de suínos do Brasil. O método de disco-difusão foi empregado usando 14 antimicrobianos: amoxicilina, penicilina, ceftiofur, ciprofloxacina, enrofloxacina, clortetraciclina, doxiciclina, oxitetraciclina, tetraciclina, eritromicina, tilmicosina, florfenicol, lincomicina e sulfadiazina/trimetoprim. Streptococcus suis e Bordetella bronchiseptica foram os patógenos que apresentaram maiores níveis de resistência. Haemophilus parasuis apresentou altos níveis de resistência à sulfadiazina/trimetoprim (9/18=50%). Observou-se que isolados das regiões Centro-Oeste e Sul apresentaram quatro vezes mais chance de serem multirresistentes do que os isolados da região Sudeste. A maioria foi resistente a pelo menos um agente antimicrobiano (98,75%; 158/160) e 31,25% (50/160) das estirpes isoladas eram multirresistentes. No geral, os resultados do presente estudo mostraram grande nível de resistência à lincomicina, eritromicina, sulfadiazina/trimetoprim e tetraciclina entre patógenos respiratórios bacterianos isolados de suínos no Brasil. Os altos níveis de resistência antimicrobiana em patógenos bacterianos respiratórios em suínos reforçam a necessidade do uso criterioso de antimicrobianos na suinocultura brasileira.(AU)

Bordetella bronchiseptica antigen enhances the production of Mycoplasma hyopneumoniae antigen-specific immunoglobulin G in mice

We previously demonstrated that Bordetella (B.) bronchiseptica antigen (Ag) showed high immunostimulatory effects on mouse bone marrow cells (BMs) while Mycoplasma (M.) hyopneumoniae Ag showed low effects. The focus of this study was to determine if B. bronchiseptica Ag can enhance the M. hyopneumoniae Ag-specific immune response and whether the host's immune system can recognize both Ags. MTT assay results revealed that each or both Ags did not significantly change BM metabolic activity. Flow cytometry analysis using carboxyfluorescein succinimidyl ester showed that B. bronchiseptica Ag can promote the division of BMs. In cytokine and nitric oxide (NO) assays, B. bronchiseptica Ag boosted production of tumor necrosis factor-alpha in M. hyopneumoniae Ag-treated BMs, and combined treatment with both Ags elevated the level of NO in BMs compared to that from treatment of M. hyopneumoniae Ag alone. Immunoglobulin (Ig)G enzyme-linked immunosorbent assay using the sera of Ag-injected mice clearly indicated that B. bronchiseptica Ag can increase the production of M. hyopneumoniae Ag-specific IgG. This study provided information valuable in the development of M. hyopneumoniae vaccines and showed that B. bronchiseptica Ag can be used both as a vaccine adjuvant and as a vaccine Ag.

Pneumonia caused by Bordetella bronchiseptica in two HIV-positive patients / Pneumonia causada por Bordetella bronchiseptica em dois pacientes HIV-positivos

ABSTRACT: CONTEXT AND OBJECTIVE: Bordetella bronchiseptica (BB) is a Gram-negative coccobacillus responsible for respiratory diseases in dogs, cats and rabbits. Reports on its development in humans are rare. However, in immunosuppressed patients, especially in those with the immunodeficiency virus (HIV), BB can cause severe pulmonary infections. We report on two cases of pneumonia caused by BB in HIV-positive male patients in a university hospital. CASE REPORT: The first case comprised a 43-year-old patient who was admitted presenting chronic leg pain and coughing, with suspected pneumonia. BB was isolated from sputum culture and was successfully treated with trimethoprim/sulfamethoxazole in association with levofloxacin. The second case comprised a 49-year-old patient who was admitted presenting fever, nausea, sweating and a dry cough, also with suspected pneumonia. BB was isolated from sputum culture, tracheal secretions and bronchoalveolar lavage. The disease was treated with ciprofloxacin but the patient died. CONCLUSION: BB should be included in the etiology of pneumonia in immunodeficient HIV patients. As far as we know, these two were the first cases of pneumonia due to BB to occur in this university hospital.

RESUMO CONTEXTO E OBJETIVO: Bordetella bronchiseptica (BB) é um cocobacilo Gram-negativo responsável por causar doenças no trato respiratório de cães, gatos e coelhos. São raros os relatos do desenvolvimento desse microrganismo em seres humanos. Porém, em pacientes imunodeprimidos, especialmente nos portadores do vírus da imunodeficiência humana (HIV), a BB pode causar infecções pulmonares graves. Nós relatamos dois casos de pneumonia por BB em pacientes do sexo masculino, HIV-positivos em um hospital universitário. RELATO DE CASO: No primeiro caso, o paciente de 43 anos foi internado apresentando dor crônica nos membros inferiores e tosse com suspeita de pneumonia. Na cultura de escarro, foi isolado BB, e a infecção foi tratada com sucesso com a associação de sulfametoxazol/trimetroprima e levofloxacino. No segundo caso, o paciente de 49 anos foi internado apresentando febre, náuseas, sudorese e tosse seca, também com suspeita de pneumonia. Das culturas de escarro, secreção traqueal e lavado bronco-alveolar, foi isolado BB, infecção tratada com ciprofloxacino: porém, o paciente foi a óbito. CONCLUSÃO: BB deve ser incluído na etiologia de pneumonia em pacientes imunocomprometidos com HIV. Pelo que é de nosso conhecimento, estes dois relatos foram os primeiros casos de pneumonia por BB que ocorreram neste hospital universitário.

Bordetella bronchiseptica Respiratory Infection in the Immunosuppressed Patient / 대한내과학회지

Bordetella bronchiseptica is a common cause of respiratory disease in animals but is a rare cause of human infection. Furthermore, most patients with Bordetella bronchiseptica infections are immunocompromised. The Bordetella bronchiseptica organism can cause pneumonia, septicemia, and peritonitis in humans with impaired immune systems. Additionally, it can lead to a life-threatening infection patients who have an underlying debilitation or impaired immunity. The respiratory tract is the most common site of infection. Sixty-two human cases of Bordetella bronchiseptica have been published in the English literature, and 84 % hadof the cases were associated with pneumonia or bronchitis. However, only one case of Bordetella bronchiseptica has been reported in South Korea, and it was associated with peritonitis. In the current study, we report a case of Bordetella bronchiseptica pneumonia diagnosed in an immunocompromised patient.

Persistent Bordetella petrii Infection Related to Bone Fractures

No abstract available.

Bacteriemia recurrente por Bordetella bronchiseptica en un paciente con trasplante de medula ósea / Bordetella bronchiseptica recurrent bacteraemia in a patient with bone marrow transplantation

Se reporta un caso de bacteriemia recurrente por Bordetella bronchiseptica en un paciente inmunocomprometido con antecedentes de trasplante alogénico de medula ósea por síndrome mielodisplásico, quien ingresó al hospital por síndrome febril. Bordetella bronchiseptica es un agente patógeno veterinario poco común en humanos que afecta principalmente a pacientes inmunocomprometidos y es causa poco frecuente de bacteriemia.

We report a case of recurrent bacteraemia caused by Bordetella bronchiseptica in an immunocompromised patient with a history of allogenic bone marrow transplantation for myelodysplastic syndrome, who was admitted to hospital with febrile syndrome. Bordetella bronchiseptica is an uncommon human pathogen which mainly affects immunocompromised patients, being a rare cause of bacteraemia.

Evaluation of adjuvant effects of fucoidan for improving vaccine efficacy

Fucoidan is a sulfated polysaccharide derived from brown seaweed, including Fucus vesiculosus. This compound is known to have immunostimulatory effects on various types of immune cells including macrophages and dendritic cells. A recent study described the application of fucoidan as a vaccine adjuvant. Vaccination is regarded as the most efficient prophylactic method for preventing harmful or epidemic diseases. To increase vaccine efficacy, effective adjuvants are needed. In the present study, we determined whether fucoidan can function as an adjuvant using vaccine antigens. Flow cytometric analysis revealed that fucoidan increases the expression of the activation markers major histocompatibility complex class II, cluster of differentiation (CD)25, and CD69 in spleen cells. In combination with Bordetella bronchiseptica antigen, fucoidan increased the viability and tumor necrosis factor-alpha production of spleen cells. Furthermore, fucoidan increased the in vivo production of antigen-specific antibodies in mice inoculated with Mycoplasma hyopneumoniae antigen. Overall, this study has provided valuable information about the use of fucoidan as a vaccine adjuvant.

Stimulatory effects of Bordetella bronchiseptica antigen on bone marrow cells and immune memory responses / 대한수의학회지

Bone marrow is a hematological and immunological organ that provides multiple immune cells, including B lymphocytes, and thus plays a critical role in the efficacy of vaccine. We previously demonstrated that Bordetella (B.) bronchiseptica antigen has high immunogenicity in spleen cells, a peripheral immune organ. In this study, we investigated the immunogenicity of B. bronchiseptica antigen in bone marrow cells, a central immune organ. B. bronchiseptica antigen increased the cellular activity of bone marrow cells and significantly enhanced the production of nitric oxide, IL-6, and TNF-alpha. Bone marrow cells primed with B. bronchiseptica antigen in vivo were harvested and stimulated with the same antigen in vitro. The stimulation of B. bronchiseptica antigen significantly increased the cellular activity and proliferation rate of the primed cells. B. bronchiseptica antigen also greatly induced the production of antigen-specific antibody in the primed cells. Taken together, the present study demonstrated that B. bronchiseptica antigen can stimulate bone marrow cells, a central immune organ, and recall the immune response of the primed bone marrow cells.

Evaluation of the immunogenicity of Bordetella bronchiseptica, a vaccine antigen / 대한수의학회지

Bordetella (B.) bronchiseptica is a causative agent of swine atrophic rhinitis that promotes colonization of the mucous membrane of the swine nasal cavity by Pasteurella (P.) multocida. Mixed infection with B. bronchiseptica and P. multocida leads to growth inhibition of pigs, resulting in significant economic loss. There are many commercial vaccines for atrophic rhinitis, including B. bronchiseptica as a killed vaccine antigen (Ag). However, the immunogenicity of killed B. bronchiseptica Ag has not yet been elucidated; therefore, this study was conducted to investigate the immunogenicity of killed B. bronchiseptica Ag and the type of immune response it induces. In vitro assays using mouse spleen cells and flow cytometry revealed that B. bronchiseptica Ag induced high proliferation capability of lymphocytes, especially B lymphocytes, and the proliferating cells showed a significant response to interleukin (IL)-2. B. bronchiseptica Ag also enhanced the production of IL-12, a representative cytokine for cell-mediated immunity. In vivo experiments using mice showed that the injection of B. bronchiseptica Ag markedly induced Ag-specific antibody. Taken together, these results indicate that B. bronchiseptica Ag has high immunogenicity by itself.

Rapid and sensitive detection of Bordetella bronchiseptica by loop-mediated isothermal amplification (LAMP)

Bordetella bronchiseptica causes acute and chronic respiratory infections in diverse animal species and occasionally in humans. In this study, we described the establishment of a simple, sensitive and cost-efficient loop-mediated isothermal amplification (LAMP) assay for the detection of B. bronchiseptica. A set of primers towards a 235 bp region within the flagellum gene of B. bronchiseptica was designed with online software.. The specificity of the LAMP assay was examined by using 6 porcine pathogens and 100 nasal swabs collected from healthy pigs and suspect infected pigs. The results indicated that positive reactions were confirmed for all B. bronchiseptica and no cross-reactivity was observed from other non-B. bronchiseptica. In sensitivity evaluations, the technique successfully detected a serial dilutions of extracted B. bronchiseptica DNA with a detection limit of 9 copies, which was 10 times more sensitive than that of PCR. Compared with conventional PCR, the higher sensitivity of LAMP method and no need for the complex instrumentation make this LAMP assay a promising alternative for the diagnosis of B. bronchiseptica in rural areas and developing countries where there lacks of complex laboratory services.

Desarrollo y validación de una PCR múltiple para el diagnóstico de Bordetella spp / Development and validation of a multiplex PCR for diagnosis of Bordetella spp / Desenvolvimento e validação de uma PCR múltipla para o diagnóstico de Bordetella spp

El objetivo del trabajo consistió en diseñar y validar una PCR en formato convencional que permita confirmar la presencia o ausencia de Bordetella pertussis y detectar otras especies del género, como Bordetella parapertussis y Bordetella bronchiseptica, que pudieran estar involucradas en el cuadro clínico de coqueluche. A tal fin se diseñó una reacción en cadena de la polimerasa (PCR) múltiple que amplifica una secuencia del promotor del gen de Toxina Pertussis y otra del gen de la Toxina Adenilato Ciclasa-Hemolisina. Se validó la metodología siguiendo esquemas publicados anteriormente. Se optimizaron las condiciones de la PCR. Se validó la metodología obteniéndose un límite de detección para ambas secuencias de 0,5 bacterias por reacción. Se validó, además, la especificidad y robustez de la técnica. Se presenta una nueva herramienta diagnóstica optimizada y validada, que permite detectar la presencia de las especies de Bordetella más frecuentemente involucradas en el cuadro clínico de coqueluche. Su uso combinado con alguna de las PCR habituales en diagnóstico, como la PCR IS481, permite aumentar la sensibilidad del diagnóstico de esta en­fermedad, la especificidad del mismo discriminando los resultados falsos positivos/negativos y aumentar el conocimiento sobre los agentes etiológicos implicados en esta patología.

The aim of the present work was to design and validate a conventional PCR that enables to confirm the presence or absence of Bordetella pertussis and to detect other Bordetella species, such as Bordetella parapertussis metoand B. bronchiseptica, that may be involved in this pathology. To this aim, a multiplex PCR that amplifies a sequence of the promoter of the Pertussis Toxin gene and a sequence of the Adenylate Cyclase Toxin-Hemolysin gene were designed. The PCR was validated following previously published schemes. PCR conditions were optimized. The methodology was validated obtaining a detection limit of 0.5 bacteria per reaction, for both sequences. Specificity and robustness of the technique were also validated. A new optimized and validated tool to detect the presence of the Bordetella species most frequently responsible of pertussis was presented. The combined use with some of the usual PCR, such as IS 481, may increase the sensitivity of the diagnosis of this disease, its specificity discriminating false positive/negative results and increase awareness of the etiologic agents involved in this pathology.

O objetivo do trabalho foi desenhar e validar uma reação em cadeia da polimerase (PCR) em formato convencional que permita confirmar a presença ou ausência de Bordetella pertussis e detectar outras espécies do gênero, como Bordetella parapertussis e Bordetella bronchiseptica, que pudessem estar envolvidas no quadro clínico de coqueluche. Para tal, foi desenhada uma PCR múltipla que amplifica uma sequência do promotor do gene de Toxina Pertussis e outra do gene da Toxina Adenilato Ciclase-Hemolisina. A metodologia foi validada seguindo esquemas publicados anteriormente. Foram otimizadas as condições da PCR. Validou-se a metodologia obtendo-se um limite de detecção para ambas as sequências de 0,5 bactérias por reação. Validou-se também a especificidade e robustez da técnica. Apresenta-se uma nova ferramenta diagnóstica otimizada e validada, que permite detectar a presença das espécies de Bordetella mais frequentemente envolvidas no quadro clínico de coqueluche. Seu uso combinado com alguma das PCR habituais em diagnóstico, como a PCR IS481, permite aumentar a sensibilidade do diagnóstico desta doença, a especificidade do mesmo discriminando os resultados falsos positivos/negativos e aumentar o conhecimento sobre os agentes etiológicos envolvidos nesta patologia.

Expression and characterization of the dermonecrotic toxin gene of Bordetella bronchiseptica / 生物工程学报

Dermonecrotic toxin (DNT) is identified as one of the most important virulence factor of Bordetella bronchiseptica. The complete coding sequence (4 356 bp) of the dnt gene was cloned into the prokaryotic expression vector pET-28a, and expressed in the Eschierichia coli BL21 (DE3) under IPTG (Isopropyl-beta-D-thiogalactopyranoside) induction. The recombinant His6-DNT protein showed immunological reactivity in the Western-blot analysis. The recombinant protein was purified from crude lysates of BL21 harboring pET-DNT with the purity of 93.2%. His6-DNT showed the dermonecrotic effects in the infant mouse assay. However, rabbit anti-serum against recombinant DNT protein could neutralize the dermonecrotic effects of native DNT to the infant mice in vivo. These findings suggest that the recombinant DNT protein retained the characteristics and immunogenicity of native DNT. Furthermore, this approach could be used to induce active immunity and serum immunoglobulin for production of a passive therapeutic reagent. In this study, we have shown that the recombinant His6-DNT protein retained the characteristics of native DNT of B. bronchiseptica, which built a good foundation for the further research on the structure and function of DNT.

Pneumonia enzoótica em javalis (Sus scrofa) / Enzootic pneumonia in wild boars (Sus scrofa)

O objetivo desse estudo foi descrever o quadro clínico e epidemiológico, os achados patológicos, bacteriológicos e imuno-histoquímicos de um surto de pneumonia em uma granja de Javalis do Distrito Federal, Brasil. Em um período de cinco meses, morreram 90 javalis. Desses, 63 tinham lesões pulmonares. Clinicamente apresentavam atraso no desenvolvimento corporal, diminuição do apetite, letargia, tosse e dificuldade respiratória, principalmente quando movimentados. Constatou-se elevação da temperatura, 40ºC em média. Na auscultação, havia crepitações e estertores pulmonares de intensidade moderada. As alterações macroscópicas nos pulmões analisados eram típicas de broncopneumonia lobular. As lesões caracterizavam-se por consolidação crânio-ventral na maioria dos pulmões. A coloração variava de difusamente vermelho-escuro a um padrão mosaico (lóbulos vermelho-escuros intercalados por lóbulos cinzas) ou difusamente acinzentados. Na maioria dos pulmões observou-se exsudato mucopurulento na luz dos brônquios e fluindo do parênquima. Histologicamente, as alterações eram de broncopneumonia purulenta e histiocitária com focos de necrose. Em alguns animais havia também hiperplasia do BALT e, na maioria dos animais, infiltração linfocítica perivascular e peribronquial. Bordetella bronchiseptica e Streptococcus spp. foram as principais bactérias isoladas. A imuno-histoquímica demonstrou a bactéria Mycoplasma hyopneumoniae no epitélio bronquiolar e bronquial e o DNA desta bactéria foi detectado pela PCR. Este é o primeiro relato de broncopneumonia em Javalis associado à infecção por M. hyopneumoniae.

The aim of this paper is to describe the clinical, epidemiological, pathological, bacteriological and immunohistochemical aspects of a pneumonia outbreak in a wild pig farm in the Distrito Federal, Brazil. Ninety wild pigs died in a period of five months, and 63 of these had pulmonary lesions. Clinically, the pigs presented reduced growth rate, anorexia, lethargy, cough and dyspnea, especially after they were moved. High body temperature (40ºC in average) was verified in some animals. Auscultation revealed moderate pulmonary crepitation and stertors. Pulmonary gross lesions were typical of lobular bronchopneumonia. Lung lesions were characterized by ventral-cranial consolidation in the majority of the cases. The color of affected pulmonary areas varied from diffuse dark red to mosaic pattern (dark red lobule intercalate by grayish lobule) or diffusely grayish. The majority of the lungs had mucopurulent exsudate in the bronchial lumen that also drained from the parenchyma cut surface. Upon microscopy, the changes were characterized by purulent and histiocytic bronchopneumonia with necrotic foci. In some animals, there was BALT hyperplasia associated with perivascular and peribronchial plasma cells and lymphocytes infiltration in most of these cases. Bordetella bronchiseptica and Streptococcus spp. were the most frequently isolated bacteria. Immunohistochemistry evaluation demonstrated Mycoplasma hyopneumoniae on the luminal surface of bronchial and bronchiolar epithelial cells, and the DNA of bacteria was detected by PCR. This is the first report of bronchopneumonia in wild boars associated with M. hyopneumoniae infection.

Relapsing Peritonitis Caused by Bordetella bronchiseptica in Continuous Ambulatory Peritoneal Dialysis Patient: A Case Report

Bordetella (B) bronchiseptica is a common veterinary pathogen, but has rarely been implicated in human infections. Most patients with B. bronchiseptica infections are compromised clinically such as in patients with a malignancy, AIDS, malnutrition, or chronic renal failure. We experienced a case of relapsing peritonitis caused by B. bronchiseptica associated with continuous ambulatory peritoneal dialysis (CAPD). A 56-yr-old male, treated with CAPD due to end stage renal disease (ESRD), was admitted with complaints of abdominal pain and a turbid peritoneal dialysate. The culture of peritoneal dialysate identified B. bronchiseptica. The patient was treated with a combination of intraperitoneal antibiotics. There were two further episodes of relapsing peritonitis, although the organism was sensitive to the used antibiotics. Finally, the indwelling CAPD catheter was removed and the patient was started on hemodialysis. This is the first report of a B. bronchiseptica human infection in the Korean literature.

Discriminative PCR of Bordetella pertussis from closely related Bordetella species using 16S rDNA Gene

BACKGROUND: Polymerase-chain reaction (PCR) detection is useful to diagnosis of pertussis at initial stage because the growth rate of Bordetella pertussis (B. pertussis) is relatively slow. Currently, the primer set for the insertion sequence IS481 (BP primer) is used widely for PCR detection of B. pertussis. However, the cross-reactivity of BP primer set with Bordetella holmesii (B. holmesii) was reported recently. Therefore, discrimination of B. pertussis and B. holmesii is needed in PCR step. For this reason, we developed new primer sets based on 16S rDNA sequence for diagnostic use and estimated the efficiency of these new primer sets. MATERIALS AND METHODS: The specific PCR primers were designed from the aligned sequence matrix of 16S rDNA genes of various Bordetella species. The specificity of designed primers were estimated using clinically important 4 Bordetella species, B. pertussis, B. holmesii, Bordetella parapertussis (B. parapertussis) and Bordetella bronchiseptica (B. bronchiseptica). The sensitivity to B. pertussis of designed primers was also estimated and compared with BP primer set. RESULTS: As the results, the developed new primer set successfully distinguished B. pertussis and other Bordetella species containing B. holmesii. In the sensitivity assay, the detectable limits of 16S-F2/16S-R1 primer set for B. pertussis were revealed as 5 pg of genomic DNA and 105 cells/mL of cell suspension. In addition to these, identical results between BP with primer and new primer were obtained in clinical samples. CONCLUSION: In this study, the specific primer set for B. pertussis was developed based on 16S rDNA sequence and this primer set did not show cross-reactivity to B. holmesii. In addition to these, the applicability of this primer set to the clinical specimens was also confirmed.

Discriminative PCR of Bordetella pertussis from closely related Bordetella species using 16S rDNA Gene

BACKGROUND: Polymerase-chain reaction (PCR) detection is useful to diagnosis of pertussis at initial stage because the growth rate of Bordetella pertussis (B. pertussis) is relatively slow. Currently, the primer set for the insertion sequence IS481 (BP primer) is used widely for PCR detection of B. pertussis. However, the cross-reactivity of BP primer set with Bordetella holmesii (B. holmesii) was reported recently. Therefore, discrimination of B. pertussis and B. holmesii is needed in PCR step. For this reason, we developed new primer sets based on 16S rDNA sequence for diagnostic use and estimated the efficiency of these new primer sets. MATERIALS AND METHODS: The specific PCR primers were designed from the aligned sequence matrix of 16S rDNA genes of various Bordetella species. The specificity of designed primers were estimated using clinically important 4 Bordetella species, B. pertussis, B. holmesii, Bordetella parapertussis (B. parapertussis) and Bordetella bronchiseptica (B. bronchiseptica). The sensitivity to B. pertussis of designed primers was also estimated and compared with BP primer set. RESULTS: As the results, the developed new primer set successfully distinguished B. pertussis and other Bordetella species containing B. holmesii. In the sensitivity assay, the detectable limits of 16S-F2/16S-R1 primer set for B. pertussis were revealed as 5 pg of genomic DNA and 105 cells/mL of cell suspension. In addition to these, identical results between BP with primer and new primer were obtained in clinical samples. CONCLUSION: In this study, the specific primer set for B. pertussis was developed based on 16S rDNA sequence and this primer set did not show cross-reactivity to B. holmesii. In addition to these, the applicability of this primer set to the clinical specimens was also confirmed.

Bordetella Bronchiseptica aislada en paciente con fibrosis quística / Isolation Bordetella bronchiseptica in a patients with cystic fibrosis

Presentamos el caso de una paciente de 15 años con Fibrosis Quística (FQ) en la cual, en dos oportunidades y con un intervalo de 2 años; se aisló Bordetella Bronchiseptica con idéntico perfil genético estudiado por electroforesis de campo pulsado. El mecanismo lesional de B. Bronchiseptica en el árbol bronquial de pacientes con FQ no esta claramente establecido, pero la habilidad de esta bacteria para inhibir la función de los leucocitos y su capacidad de adherirse a las células del epitelio bronquial explicaría su capacidad infectiva y su persistencia en el tracto respiratorio.

Pseudooutbreak of Acinetobacter spp. Bacteriuria Confirmed by 16S rRNA Gene Sequence Analysis

BACKGROUND: Acinetobacter spp. is increasingly implicated in hospital-acquired infections. We experienced a pseudooutbreak of Bordetella bronchiseptica bacteriuria identified with biochemical tests, that was later identified as Acinetobacter spp. by using 16S rRNA gene sequence analysis. MATERIALS AND METHODS: Five in-ward patients were found to have B. bronchiseptica bacteriuria without symptoms of urinary tract infection between September 23 and 26 of 2005. We conducted pulsed field gel electrophoresis (PFGE) of the bacteria and epidemiological investigation of this pseudooutbreak. In addition, 16S rRNA gene sequence analysis was performed for the verification of the strains. RESULTS: All 5 isolates were identified as B. bronchiseptica with similar antibiogram by VITEK system. There was no evidence of any symptom or sign of urinary tract infection. The source of this pseudooutbreak was not detected even after performing environmental culture and interviews with healthcare workers. We could not get the appropriate results from the first PFGE with XbaI restriction enzyme. B. bronchiseptica is an unusual organism in human so we conducted 16S rRNA gene sequence analysis for verification. The analysis of 16S rRNA gene sequence with 5 isolates demonstrated 99-100% similarity to a sequence of Acinetobacter spp. (AU1523). According to the results of 16S rRNA gene sequence analysis, we performed the second PFGE with SmaI restriction enzyme, which showed indistinguishable pattern among the all 5 isolates. CONCLUSION: This investigation suggests that the combined method of 16s rRNA gene sequence analysis and PFGE would be helpful for investigation of outbreak caused by unusual organisms

Pseudooutbreak of Acinetobacter spp. Bacteriuria Confirmed by 16S rRNA Gene Sequence Analysis

BACKGROUND: Acinetobacter spp. is increasingly implicated in hospital-acquired infections. We experienced a pseudooutbreak of Bordetella bronchiseptica bacteriuria identified with biochemical tests, that was later identified as Acinetobacter spp. by using 16S rRNA gene sequence analysis. MATERIALS AND METHODS: Five in-ward patients were found to have B. bronchiseptica bacteriuria without symptoms of urinary tract infection between September 23 and 26 of 2005. We conducted pulsed field gel electrophoresis (PFGE) of the bacteria and epidemiological investigation of this pseudooutbreak. In addition, 16S rRNA gene sequence analysis was performed for the verification of the strains. RESULTS: All 5 isolates were identified as B. bronchiseptica with similar antibiogram by VITEK system. There was no evidence of any symptom or sign of urinary tract infection. The source of this pseudooutbreak was not detected even after performing environmental culture and interviews with healthcare workers. We could not get the appropriate results from the first PFGE with XbaI restriction enzyme. B. bronchiseptica is an unusual organism in human so we conducted 16S rRNA gene sequence analysis for verification. The analysis of 16S rRNA gene sequence with 5 isolates demonstrated 99-100% similarity to a sequence of Acinetobacter spp. (AU1523). According to the results of 16S rRNA gene sequence analysis, we performed the second PFGE with SmaI restriction enzyme, which showed indistinguishable pattern among the all 5 isolates. CONCLUSION: This investigation suggests that the combined method of 16s rRNA gene sequence analysis and PFGE would be helpful for investigation of outbreak caused by unusual organisms