Effect of carbohydrates on the adhesion of Bordetella bronchiseptica to the respiratory epithelium in rabbits.

This study proposes an ecological approach for preventing respiratory tract infections caused by Bordetella bronchiseptica in mammals using a mixture of carbohydrates. In an in vivo study, 51-day-old New Zealand rabbits were treated with a solution containing 1 × 107 CFUs of B. bronchiseptica and 250 µg of one of the following carbohydrates: N acetylglucosamine (GlcNAc), N acetylgalactosamine (GalNAc), alpha methyl mannose (AmeMan), alpha methyl glucose (AmeGlc) and sialic acid (Neu5AC). Positive (B. bronchiseptica) and negative (Physiological Saline Solution (PSS)) controls were included. Animals treated with GlcNAc or AmeGlc showed no clinical signs of infection and exhibited a significant reduction (p < 0.05) in the severity of microscopic lesions evaluated in the nasal cavity and lung compared with the positive controls. Additionally, the presence of bacteria was not detected through microbiological isolation or PCR in the lungs of animals treated with these sugars. Use of a mixture of GlcNAc and AmeGlc resulted in greater inhibition of microscopic lesions, with a significant reduction (p < 0.05) in the severity of these lesions compared to the results obtained using individual sugars. Furthermore, the bacterium was not detected through microbiological isolation, Polymerase Chain Reaction (PCR) or indirect immunoperoxidase (IIP) in this group.

Neumonía por Bordetella bronchiseptica en paciente con antecedente de linfoma pulmonar / Bordetella bronchiseptica pneumonia in a patient with a history of pulmonary lymphoma

Resumen Bordetella bronchiseptica es un cocobacilo Gram negativo patógeno de animales que, con poca frecuencia causa infecciones en seres humanos. La mayoría de casos registrados en la literatura están asociados con pacientes que presentan algún tipo de inmunosupresión. Este reporte de caso se refiere a una paciente femenina de 67 años con antecedentes de linfoma pulmonar, que recibió quimioterapia y radioterapia 16 años atrás, fue ingresada al Servicio de Medicina Interna del Hospital Tomás Casas Casajús con un diagnóstico de neumonía bacteriana, tras descartarse infección por Covid19. Unos días después, se aisló una B. brochiseptica de una muestra de esputo y con el reporte de la prueba sensibilidad a los antibióticos, se modificó la terapia de antibióticos que originalmente se había indicado, lo que se conlleva a una mejoría en el estado físico de la paciente. Sin embargo, debido a una aparente infección nosocomial la paciente se contagió de SARS- CO2 y falleció debido a las complicaciones causadas por el Covid19.

Abstract Bordetella bronchiseptica is an animal pathogenic Gram negative coccobacillus that infrequently causes human infections. Most of the cases recorded in the literature are associated with patients with some type of immunosuppression. In this case, a 67-year- old female patient with a history of pulmonary lymphoma, who received chemotherapy and radiotherapy 16 years ago, is admitted to the Internal Medicine Service of the Tomás Casas Casajús Hospital, with a diagnosis of bacterial pneumonia, after ruling out Covid19 infection. A few days later, a B. brochiseptica is isolated from a sputum sample and with the report of the antibiotic sensitivity test, the antibiotic therapy that had originally been indicated is modified, which is reflected in an improvement in the physical state of the patient. However, due to an apparent nosocomial infection, the patient becomes infected with SARS-CO2 and dies due to complications caused by Covid19.

Bordetella pertussis, a reemerging pathogen in pediatric respiratory infections. A study in Quito, Ecuador.

Bordetella pertussis is the causative agent of pertussis, which mainly affects unvaccinated children, while Bordetella parapertussis causes a disease presenting clinical characteristics that are indistinguishable from whooping cough. Despite high vaccination coverage, pertussis remains a public health concern worldwide, with approximately 140000 cases reported annually. Here we determined the prevalence of B. pertussis and B. parapertussis infection among infants under one year of age by polymerase chain reaction (PCR); our aim being to identify whether the data obtained relates to the relevant sociodemographic and clinical data. The study included 86 samples of nasopharyngeal swabs from infants aged between 0-12 months, who were reported as probable cases of whooping cough by the health centers around the Ecuadorian highlands, from August 2016 to July 2017. The nasopharyngeal swabs were cultured and microbiological and molecular analyses were performed. B. pertussis was identified by PCR in 41% of the samples (30/86), more than half of which corresponded to infants aged between 0-3 months. Moreover, a statistically significant correlation (p<0.05) between the identification of bacteria in culture and the catarrhal stage of the disease was observed. The results obtained from the study highlighted the need for an active national surveillance of pertussis, in particular for laboratory testing, to provide a highly sensitive and more specific diagnosis of Bordetella infection.

Bordetella bronchiseptica Diguanylate Cyclase BdcA Regulates Motility and Is Important for the Establishment of Respiratory Infection in Mice.

Bacteria can be motile and planktonic or, alternatively, sessile and participating in the biofilm mode of growth. The transition between these lifestyles can be regulated by a second messenger, cyclic dimeric GMP (c-di-GMP). High intracellular c-di-GMP concentration correlates with biofilm formation and motility inhibition in most bacteria, including Bordetella bronchiseptica, which causes respiratory tract infections in mammals and forms biofilms in infected mice. We previously described the diguanylate cyclase BdcA as involved in c-di-GMP synthesis and motility regulation in B. bronchiseptica; here, we further describe the mechanism whereby BdcA is able to regulate motility and biofilm formation. Amino acid replacement of GGDEF with GGAAF in BdcA is consistent with the conclusion that diguanylate cyclase activity is necessary for biofilm formation and motility regulation, although we were unable to confirm the stability of the mutant protein. In the absence of the bdcA gene, B. bronchiseptica showed enhanced motility, strengthening the hypothesis that BdcA regulates motility in B. bronchiseptica We showed that c-di-GMP-mediated motility inhibition involved regulation of flagellin expression, as high c-di-GMP levels achieved by expressing BdcA significantly reduced the level of flagellin protein. We also demonstrated that protein BB2109 is necessary for BdcA activity, motility inhibition, and biofilm formation. Finally, absence of the bdcA gene affected bacterial infection, implicating BdcA-regulated functions as important for bacterium-host interactions. This work supports the role of c-di-GMP in biofilm formation and motility regulation in B. bronchiseptica, as well as its impact on pathogenesis.IMPORTANCE Pathogenesis of Bordetella spp., like that of a number of other pathogens, involves biofilm formation. Biofilms increase tolerance to biotic and abiotic factors and are proposed as reservoirs of microbes for transmission to other organs (trachea, lungs) or other hosts. Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is a second messenger that regulates transition between biofilm and planktonic lifestyles. In Bordetella bronchiseptica, high c-di-GMP levels inhibit motility and favor biofilm formation. In the present work, we characterized a B. bronchiseptica diguanylate cyclase, BdcA, which regulates motility and biofilm formation and affects the ability of B. bronchiseptica to colonize the murine respiratory tract. These results provide us with a better understanding of how B. bronchiseptica can infect a host.

Bordetella trematum infection: case report and review of previous cases.

BACKGROUND: Bordetella trematum is an infrequent Gram-negative coccobacillus, with a reservoir, pathogenesis, a life cycle and a virulence level which has been poorly elucidated and understood. Related information is scarce due to the low frequency of isolates, so it is important to add data to the literature about this microorganism. CASE PRESENTATION: We report a case of a 74-year-old female, who was referred to the hospital, presenting with ulcer and necrosis in both legs. Therapy with piperacillin-tazobactam was started and peripheral artery revascularization was performed. During the surgery, a tissue fragment was collected, where Bordetella trematum, Stenotrophomonas maltophilia, and Enterococcus faecalis were isolated. After surgery, the intubated patient was transferred to the intensive care unit (ICU), using vasoactive drugs through a central venous catheter. Piperacillin-tazobactam was replaced by meropenem, with vancomycin prescribed for 14 days. Four days later, levofloxacin was added for 24 days, aiming at the isolation of S. maltophilia from the ulcer tissue. The necrotic ulcers evolved without further complications, and the patient's clinical condition improved, leading to temporary withdrawal of vasoactive drugs and extubation. Ultimately, however, the patient's general condition worsened, and she died 58 days after hospital admission. CONCLUSIONS: Despite being a rare finding, B. trematum is typically associated with the clinical manifestation of disorders that predispose to ulcer development, which can be infected by microorganisms. The combination of antibiotic therapy and surgical debridement plays a key role in preventing systemic infections. Monitoring the appearance of new cases of B. trematum is essential, since it can be an emerging microorganism. Isolating and defining the clinical relevance of unusual bacteria yields a more accurate perspective in the development of new diagnostic tools and allows for assessment of proper antimicrobial therapy.

Outer-Membrane-Vesicle-Associated O Antigen, a Crucial Component for Protecting Against Bordetella parapertussis Infection.

Bordetella parapertussis is a respiratory-disease pathogen producing symptomatology similar to that of pertussis but of underestimated incidence and with no specific vaccine existing. We recently designed a vaccine candidate from B. parapertussis outer-membrane vesicles (OMVs) that proved to be safe and protective in a murine-infection model. Based on protection recently reported for the B. parapertussis O antigen in aqueous solution, we assessed here whether the B. parapertussis O-antigen-containing lipopolysaccharide (BppLPS-O+) embedded in the membranes, as present in B. parapertussis-derived OMVs (OMVs(Bpp-LPS-O+)), was the component responsible for that previously observed protection by OMVs. By performing a comparative study with OMVs from a human strain with undetectable O antigen (OMVs(Bpp-LPS-O-)), we demonstrated that the OMVs(Bpp-LPS-O+), but not the OMVs(Bpp-LPS-O-), protected mice against sublethal B. parapertussis infections. Indeed, the B. parapertussis loads were significantly reduced in the lungs of OMVs(Bpp-LPS-O+) -vaccinated animals, with the CFUs recovered being decreased by 4 log units below those detected in the non-immunized animals or in the animals treated with the OMVs(Bpp-LPS-O-), (p < 0.001). We detected that the OMVs(Bpp-LPS-O+) induced IgG antibodies against B. parapertussis whole-cell lysates, which immunocomponents recognized, among others, the O antigen and accordingly conferred protection against B. parapertussis infection, as observed in in-vivo-passive-transfer experiments. Of interest was that the OMVs(Bpp-LPS-O+) -generated sera had opsonophagocytic and bactericidal capabilities that were not detected with the OMVs(Bpp-LPS-O-)-induced sera, suggesting that those activities were involved in the clearance of B. parapertussis. Though stimulation of cultured spleen cells from immunized mice with formulations containing the O antigen resulted in gamma interferon (IFN-γ) and interleukin-17 production, spleen cells from OMVs(Bpp-LPS-O+) -immunized mice did not significantly contribute to the observed protection against B. parapertussis infection. The protective capability of the B. parapertussis O antigen was also detected in formulations containing both the OMVs derived from B. pertussis and purified BppLPS-O+. This combined formulation protected mice against B. pertussis along with B. parapertussis.

Membrane Vesicles Derived from Bordetella bronchiseptica: Active Constituent of a New Vaccine against Infections Caused by This Pathogen.

Bordetella bronchiseptica, a Gram-negative bacterium, causes chronic respiratory tract infections in a wide variety of mammalian hosts, including humans (albeit rarely). We recently designed Bordetella pertussis and Bordetella parapertussis experimental vaccines based on outer membrane vesicles (OMVs) derived from each pathogen, and we obtained protection against the respective infections in mice. Here, we demonstrated that OMVs derived from virulent-phase B. bronchiseptica (OMVBbvir+) protected mice against sublethal infections with different B. bronchiseptica strains, two isolated from farm animals and one isolated from a human patient. In all infections, we observed that the B. bronchiseptica loads were significantly reduced in the lungs of vaccinated animals; the lung-recovered CFU were decreased by ≥4 log units, compared with those detected in the lungs of nonimmunized animals (P < 0.001). In the OMVBbvir+-immunized mice, we detected IgG antibody titers against B. bronchiseptica whole-cell lysates, along with an immune serum having bacterial killing activity that both recognized B. bronchiseptica lipopolysaccharides and polypeptides such as GroEL and outer membrane protein C (OMPc) and demonstrated an essential protective capacity against B. bronchiseptica infection, as detected by passive in vivo transfer experiments. Stimulation of cultured splenocytes from immunized mice with OMVBbvir+ resulted in interleukin 5 (IL-5), gamma interferon (IFN-γ), and IL-17 production, indicating that the vesicles induced mixed Th2, Th1, and Th17 T-cell immune responses. We detected, by adoptive transfer assays, that spleen cells from OMVBbvir+-immunized mice also contributed to the observed protection against B. bronchiseptica infection. OMVs from avirulent-phase B. bronchiseptica and the resulting induced immune sera were also able to protect mice against B. bronchiseptica infection.IMPORTANCEBordetella bronchiseptica, a Gram-negative bacterium, causes chronic respiratory tract infections in a wide variety of mammalian hosts, including humans (albeit rarely). Several vaccines aimed at preventing B. bronchiseptica infection have been developed and used, but a safe effective vaccine is still needed. The significance and relevance of our research lie in the characterization of the OMVs derived from B. bronchiseptica as the source of a new experimental vaccine. We demonstrated here that our formulation based on OMVs derived from virulent-phase B. bronchiseptica (OMVBbvir+) was effective against infections caused by B. bronchiseptica isolates obtained from different hosts (farm animals and a human patient). In vitro and in vivo characterization of humoral and cellular immune responses induced by the OMVBbvir+ vaccine enabled a better understanding of the mechanism of protection necessary to control B. bronchiseptica infection. Here we also demonstrated that OMVs derived from B. bronchiseptica in the avirulent phase and the corresponding induced humoral immune response were able to protect mice from B. bronchiseptica infection. This realization provides the basis for the development of novel vaccines not only against the acute stages of the disease but also against stages of the disease or the infectious cycle in which avirulence factors could play a role.

A recombinant iron transport protein from Bordetella pertussis confers protection against Bordetella parapertussis.

Whooping cough, which is caused by Bordetella pertussis and B. parapertussis, is a reemerging disease. New protective antigens are needed to improve the efficacy of current vaccines against both species. Using proteomic tools, it was here found that B. parapertussis expresses a homolog of AfuA, a previously reported new vaccine candidate against B. pertussis. It was found that this homolog, named AfuABpp , is expressed during B. parapertussis infection, exposed on the surface of the bacteria and recognized by specific antibodies induced by the recombinant AfuA cloned from B. pertussis (rAfuA). Importantly, the presence of the O-antigen, a molecule that has been found to shield surface antigens on B. parapertussis, showed no influence on antibody recognition of AfuABpp on the bacterial surface. The present study further showed that antibodies induced by immunization with the recombinant protein were able to opsonize B. parapertussis and promote bacterial uptake by neutrophils. Finally, it was shown that this antigen confers protection against B. parapertussis infection in a mouse model. Altogether, these results indicate that AfuA is a good vaccine candidate for acellular vaccines protective against both causative agents of whooping cough.

Bordetella parapertussis Circumvents Neutrophil Extracellular Bactericidal Mechanisms.

B. parapertussis is a whooping cough etiological agent with the ability to evade the immune response induced by pertussis vaccines. We previously demonstrated that in the absence of opsonic antibodies B. parapertussis hampers phagocytosis by neutrophils and macrophages and, when phagocytosed, blocks intracellular killing by interfering with phagolysosomal fusion. But neutrophils can kill and/or immobilize extracellular bacteria through non-phagocytic mechanisms such as degranulation and neutrophil extracellular traps (NETs). In this study we demonstrated that B. parapertussis also has the ability to circumvent these two neutrophil extracellular bactericidal activities. The lack of neutrophil degranulation was found dependent on the O antigen that targets the bacteria to cell lipid rafts, eventually avoiding the fusion of nascent phagosomes with specific and azurophilic granules. IgG opsonization overcame this inhibition of neutrophil degranulation. We further observed that B. parapertussis did not induce NETs release in resting neutrophils and inhibited NETs formation in response to phorbol myristate acetate (PMA) stimulation by a mechanism dependent on adenylate cyclase toxin (CyaA)-mediated inhibition of reactive oxygen species (ROS) generation. Thus, B. parapertussis modulates neutrophil bactericidal activity through two different mechanisms, one related to the lack of proper NETs-inducer stimuli and the other one related to an active inhibitory mechanism. Together with previous results these data suggest that B. parapertussis has the ability to subvert the main neutrophil bactericidal functions, inhibiting efficient clearance in non-immune hosts.

Homologs of the LapD-LapG c-di-GMP Effector System Control Biofilm Formation by Bordetella bronchiseptica.

Biofilm formation is important for infection by many pathogens. Bordetella bronchiseptica causes respiratory tract infections in mammals and forms biofilm structures in nasal epithelium of infected mice. We previously demonstrated that cyclic di-GMP is involved in biofilm formation in B. bronchiseptica. In the present work, based on their previously reported function in Pseudomonas fluorescens, we identified three genes in the B. bronchiseptica genome likely involved in c-di-GMP-dependent biofilm formation: brtA, lapD and lapG. Genetic analysis confirmed a role for BrtA, LapD and LapG in biofilm formation using microtiter plate assays, as well as scanning electron and fluorescent microscopy to analyze the phenotypes of mutants lacking these proteins. In vitro and in vivo studies showed that the protease LapG of B. bronchiseptica cleaves the N-terminal domain of BrtA, as well as the LapA protein of P. fluorescens, indicating functional conservation between these species. Furthermore, while BrtA and LapG appear to have little or no impact on colonization in a mouse model of infection, a B. bronchiseptica strain lacking the LapG protease has a significantly higher rate of inducing a severe disease outcome compared to the wild type. These findings support a role for c-di-GMP acting through BrtA/LapD/LapG to modulate biofilm formation, as well as impact pathogenesis, by B. bronchiseptica.

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.

Pneumonia caused by Bordetella bronchiseptica in two HIV-positive patients.

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.

Purificación del toxoide pertúsico de Bordetella pertussis / Pertussis Toxoid Purification of Bordetella pertussis

Introducción: el toxoide pertúsico es una proteína muy utilizada en las vacunas acelulares contra la tosferina. Desarrollar un protocolo para su purificación con pocos pasos y con parámetros de purificación adecuados, es muy importante para su caracterización química y la valoración de los efectos adversos que puede provocar al inocular principalmente a ratones. Los protocolos de purificación descritos en la literatura científica involucran varios pasos de purificación y bajos rendimientos.Objetivo: desarrollar un protocolo de purificación del toxoide pertúsico que garantice un grado de pureza alto y un rendimiento de más de 80% a partir de cultivos de un mutante de B. pertussis obtenido en el Centro Nacional de Investigaciones Científicas (CNIC).Métodos: para purificar el toxoide se usó una columna de intercambio catiónico Fractogel EMD SO-3 y se eluyó incrementando el pH y la concentración salina por pasos hasta obtener los resultados esperados. La determinación de proteínas totales se realizó mediante el micrométodo linealizado de Bradfor10 usando como patrón albúmina de suero bovina (BSA).Resultados: se obtuvo 3,28 mg de toxoide por cada litro de cultivo con un rendimiento de 86,7% y un grado de pureza de 96,7%.Conclusiones: el procedimiento de purificación descrito permite obtener el toxoide pertúsico con un rendimiento mayor que el 80% y alto grado de pureza, lo que garantiza la calidad requerida para su caracterización bioquímica e inmunológica.(AU)

Introduction: pertussis toxoid is a widely used in acellular vaccines against pertussis protein. Develop a protocol for purification with few steps and with parameters suitable purification is very important for chemical characterization and assessment of adverse effects that can lead to inoculate mainly mice. Purification protocols described in scientific literature involve several purification steps and low yields.Objective: to develop a protocol for purification of pertussis toxoid to ensure a high degree of purity and a yield of over 80% from cultures of a mutant of B. pertussis obtained at the National Center for Scientific Research (CNIC).Methods: for purifying the toxoid cation a Fractogel EMD SO-3 cationic interchange column was used by increasing the pH and salt concentration to obtain the expected results. The total protein determination was performed using linearized macromethod of Bradfor10 using as bovine serum albumin standard (BSA). Results: 3.28 mg toxoid was obtained per liter of culture with a yield higher than 86.7% and a purity of 96.7%.Conclusions: the purification process described allows for the pertussis toxoid with a higher yield than 80% and high purity, ensuring the quality required for biochemical and immunological characterization.

Adenovirus 2, Bordetella bronchiseptica, and Parainfluenza Molecular Diagnostic Assay Results in Puppies After vaccination with Modified Live Vaccines.

BACKGROUND: Canine adenovirus 2, parainfluenza, and Bordetella bronchiseptica cause respiratory disease in dogs, and each has a modified live intranasal vaccine available. Molecular diagnostic assays to amplify specific nucleic acids are available for each of these agents. If positive molecular diagnostic assay results are common after vaccination, the positive predictive value of the diagnostic assays for disease would be decreased. OBJECTIVE: To determine the impact of administration of commercially available modified live topical adenovirus 2, B. bronchiseptica, and parainfluenza vaccine has on the results of a commercially available PCR panel. ANIMALS: Eight puppies from a research breeding facility negative for these pathogens. METHODS: Blinded prospective pilot study. Puppies were vaccinated with a single dose of modified live topical adenovirus 2, B. bronchiseptica, and parainfluenza and parenteral dose of adenovirus 2, canine distemper virus, and parvovirus. Nasal and pharyngeal swabs were collected on multiple days and submitted for PCR assay. RESULTS: Nucleic acids of all 3 organisms contained in the topical vaccine were detected from both samples multiple times through 28 days after vaccination with higher numbers of positive samples detected between days 3 and 10 after vaccination. CONCLUSIONS AND CLINICAL IMPORTANCE: Vaccine status should be considered when interpreting respiratory agent PCR results if modified live vaccines have been used. Development of quantitative PCR and wild-type sequencing are necessary to improve positive predictive value of these assays by distinguishing vaccinate from natural infection.

Bordetella biofilms: a lifestyle leading to persistent infections.

Bordetella bronchiseptica and B. pertussis are Gram-negative bacteria that cause respiratory diseases in animals and humans. The current incidence of whooping cough or pertussis caused by B. pertussis has reached levels not observed since the 1950s. Although pertussis is traditionally known as an acute childhood disease, it has recently resurged in vaccinated adolescents and adults. These individuals often become silent carriers, facilitating bacterial circulation and transmission. Similarly, vaccinated and non-vaccinated animals continue to be carriers of B. bronchiseptica and shed bacteria resulting in disease outbreaks. The persistence mechanisms of these bacteria remain poorly characterized. It has been proposed that adoption of a biofilm lifestyle allows persistent colonization of the mammalian respiratory tract. The history of Bordetella biofilm research is only a decade long and there is no single review article that has exclusively focused on this area. We systematically discuss the role of Bordetella factors in biofilm development in vitro and in the mouse respiratory tract. We further outline the implications of biofilms to bacterial persistence and transmission in humans and for the design of new acellular pertussis vaccines.

Outer membrane protein OmpQ of Bordetella bronchiseptica is required for mature biofilm formation.

Bordetella bronchiseptica, an aerobic Gram-negative bacterium, is capable of colonizing the respiratory tract of diverse animals and chronically persists inside the hosts by forming biofilm. Most known virulence factors in Bordetella species are regulated by the BvgAS two-component transduction system. The Bvg-activated proteins play a critical role during host infection. OmpQ is an outer membrane porin protein which is expressed under BvgAS control. Here, we studied the contribution of OmpQ to the biofilm formation process by B. bronchiseptica. We found that the lack of expression of OmpQ did not affect the growth kinetics and final biomass of B. bronchiseptica under planktonic growth conditions. The ΔompQ mutant strain displayed no differences in attachment level and in early steps of biofilm formation. However, deletion of the ompQ gene attenuated the ability of B. bronchiseptica to form a mature biofilm. Analysis of ompQ gene expression during the biofilm formation process by B. bronchiseptica showed a dynamic expression pattern, with an increase of biofilm culture at 48 h. Moreover, we demonstrated that the addition of serum anti-OmpQ had the potential to reduce the biofilm biomass formation in a dose-dependent manner. In conclusion, we showed for the first time, to the best of our knowledge, evidence of the contribution of OmpQ to a process of importance for B. bronchiseptica pathobiology. Our results indicate that OmpQ plays a role during the biofilm development process, particularly at later stages of development, and that this porin could be a potential target for strategies of biofilm formation inhibition.

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.

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

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.

Clinical, laboratorial and radiographic predictors of Bordetella pertussis infection / Preditores clínicos, laboratoriais e radiográficos para infecção por Bordetella pertussis

OBJECTIVE: To identify clinical, laboratorial and radiographic predictors for Bordetella pertussis infection. METHODS: This was a retrospective study, which analyzed medical records of all patients submitted to a molecular dignosis (qPCR) for B. pertussis from September 2011 to January 2013. Clinical and laboratorial data were reviewed, including information about age, sex, signs/symptoms, length of hospitalization, blood cell counts, imaging findings, coinfection with other respiratory pathogens and clinical outcome. RESULTS: 222 cases were revised. Of these, 72.5% had proven pertussis, and 60.9% were under 1 year old. In patients aging up to six months, independent predictors for B. pertussis infection were (OR 8.0, CI 95% 1.8-36.3; p=0.007) and lymphocyte count >104/µL (OR 10.0, CI 95% 1.8-54.5; p=0.008). No independent predictors of B. pertussis infection could be determined for patients older than six months. Co-infection was found in 21.4% of patients, of which 72.7% were up to six months of age. Adenovirus was the most common agent (40.9%). In these patients, we were not able to identify any clinical features to detect patients presenting with a respiratory co-infection, even though longer hospital stay was observed in patients with co-infections (12 vs. 6 days; p=0.009). CONCLUSIONS: Cyanosis and lymphocytosis are independent predictors for pertussis in children up to 6 months old...

OBJETIVO: Identificar preditores clínicos, laboratoriais e radiológicos da infecção por Bordetella pertussis. MÉTODOS: Trabalho retrospectivo, com análise de prontuários clínicos de todos os indivíduos submetidos ao diagnóstico molecular (qPCR) para B. pertussis de setembro de 2011 à janeiro de 2013. Foram revistos dados clínicos e laboratoriais, incluindo informações sobre idade, sexo, sinais/sintomas, tempo de hospitalização, contagens de células sanguíneas, exames de imagem, co-infecção com outros patógenos respiratórios, e evolução clínica. RESULTADOS: 222 casos foram revistos, do quais 72,5% tinham coqueluche confirmada, sendo 60,9% menores de um ano de idade. Foram observados preditores independentes para B. pertussis em pacientes com menos de seis meses de idade. Nesses casos, os preditores identificados foram cianose (OR 8,0; CI 95% 1,8-36,3; p=0,007) e contagem de linfócitos >104/µL (OR 10,0, CI 95% 1,8-54,5; p=0,008). Preditores de coqueluche não puderam ser determinados para crianças maiores de 6 meses de idade. Coinfecção foi encontrada em 21,4% dos pacientes, dos quais 72,7% tinham até seis meses de idade, sendo que o adenovírus foi o agente mais comum (40,9%). Nesses indivíduos, não foram observadas características clíncias capazes de distinguir pacientes com co-infecção, porém foi verificado um maior tempo de internação hospitalar nos pacientes com mais de um agente infeccioso detectado (12 vs. 6 dias; p=0,009). CONCLUSÕES: Cianose e linfocitose são preditores independentes para coqueluche em crianças com até seis meses de idade...