Estudio piloto para determinar la asociación entre la microbiota intestinal y la fractura de cadera por fragilidad / Pilot study to determine the association between gut microbiota and fragility hip fracture

Introducción: Las fracturas de cadera son la causa más frecuente de ingreso hospitalario en los servicios de ortopedia de Europa y suponen un importante problema sanitario. Por ello, es de gran interés identificar factores de riesgo adicionales que nos ayuden a comprender mejor la fisiopatología de estas fracturas y a mejorar nuestra capacidad preventiva. Existen datos suficientes para apoyar la teoría de la modulación de la masa ósea por la microbiota intestinal (osteomicrobiología); sin embargo, faltan estudios clínicos en humanos que relacionen directamente la microbiota con el riesgo de fractura de cadera. Material y métodos: Estudio observacional, analítico, de casos y controles. La muestra consta de 50 pacientes y se distribuye de la siguiente manera: 25 pacientes ancianos con fractura de cadera por fragilidad y 25 controles sanos sin fractura. Se analizó la microbiota intestinal mediante extracción de ADN de muestras de heces y secuenciación del ADN ribosómico 16S tras la generación de bibliotecas de genes. Resultados: La diversidad alfa reveló una elevación de los estimadores para el nivel taxonómico de clase en el grupo de fracturas de cadera. Los órdenes Bacteroidales, Oscillospirales, Lachnospirales, Peptostreptococcales-Tissierellales y Enterobacterales fueron los órdenes dominantes en ambos grupos. En los pacientes con fractura, se observó un aumento porcentual significativo del orden de Bacteroidales (p<0,001) y Peptostreptococcales-Tissierellales (p<0,005), así como una disminución de las del orden Lachnospirales (p<0,001) respecto a los controles. Conclusiones:Este estudio ha encontrado una asociación entre una microbiota específica en pacientes ancianos con fractura de cadera por fragilidad. Estos hallazgos abren la puerta a nuevas estrategias para prevenir las fracturas de cadera. Es posible que la modificación de la microbiota mediante probióticos se revele como un método eficaz para reducir el riesgo de fractura de cadera.(AU)

Introduction: Hip fractures are the most common cause of hospital admission to orthopaedic departments in Europe and they generate a major health problem. Therefore, it is of great interest to identify additional risk factors that will help us to better understand the pathophysiology of these fractures and improve our preventive capacity. There is sufficient data to support the theory of modulation of bone mass by gut microbiota (osteomicrobiology); however, there is a lack of human clinical studies directly linking microbiota to hip fracture risk. Material and methods: Observational, analytical, case–control study. The sample consisted of 50 patients and it was distributed as follows: 25 elderly patients with fragility hip fracture and 25 subjects without fracture. The intestinal microbiota was determined by DNA extraction from stool samples and 16S ribosomal DNA sequencing after generation of gene libraries. Results: Alpha diversity revealed an elevation of the estimators for the taxonomic class level in the hip fracture group. The orders Bacteroidales, Oscillospirales, Lachnospirales, Peptostreptococcales-Tissierellales and Enterobacterales were the dominant orders in both groups. In patients with fracture, a significant percentage increase in the orders Bacteroidales (p<.001) and Peptostreptococcales-Tissierellales (p<.005) was observed, as well as a decrease in the orders Lachnospirales (p<.001) compared to controls. Conclusions: This study has found an association between a specific microbiota in elderly patients with fragility hip fracture. These findings open the door to new strategies to prevent hip fractures. Modification of the microbiota through probiotics may prove to be an effective method to reduce the risk of hip fracture.(AU)

[Translated article] Pilot study to determine the association between gut microbiota and fragility hip fracture / Estudio piloto para determinar la asociación entre la microbiota intestinal y la fractura de cadera por fragilidad

Introducción: Las fracturas de cadera son la causa más frecuente de ingreso hospitalario en los servicios de ortopedia de Europa y suponen un importante problema sanitario. Por ello, es de gran interés identificar factores de riesgo adicionales que nos ayuden a comprender mejor la fisiopatología de estas fracturas y a mejorar nuestra capacidad preventiva. Existen datos suficientes para apoyar la teoría de la modulación de la masa ósea por la microbiota intestinal (osteomicrobiología); sin embargo, faltan estudios clínicos en humanos que relacionen directamente la microbiota con el riesgo de fractura de cadera. Material y métodos: Estudio observacional, analítico, de casos y controles. La muestra consta de 50 pacientes y se distribuye de la siguiente manera: 25 pacientes ancianos con fractura de cadera por fragilidad y 25 controles sanos sin fractura. Se analizó la microbiota intestinal mediante extracción de ADN de muestras de heces y secuenciación del ADN ribosómico 16S tras la generación de bibliotecas de genes. Resultados: La diversidad alfa reveló una elevación de los estimadores para el nivel taxonómico de clase en el grupo de fracturas de cadera. Los órdenes Bacteroidales, Oscillospirales, Lachnospirales, Peptostreptococcales-Tissierellales y Enterobacterales fueron los órdenes dominantes en ambos grupos. En los pacientes con fractura, se observó un aumento porcentual significativo del orden de Bacteroidales (p<0,001) y Peptostreptococcales-Tissierellales (p<0,005), así como una disminución de las del orden Lachnospirales (p<0,001) respecto a los controles. Conclusiones:Este estudio ha encontrado una asociación entre una microbiota específica en pacientes ancianos con fractura de cadera por fragilidad. Estos hallazgos abren la puerta a nuevas estrategias para prevenir las fracturas de cadera. Es posible que la modificación de la microbiota mediante probióticos se revele como un método eficaz para reducir el riesgo de fractura de cadera.(AU)

Introduction: Hip fractures are the most common cause of hospital admission to orthopaedic departments in Europe and they generate a major health problem. Therefore, it is of great interest to identify additional risk factors that will help us to better understand the pathophysiology of these fractures and improve our preventive capacity. There is sufficient data to support the theory of modulation of bone mass by gut microbiota (osteomicrobiology); however, there is a lack of human clinical studies directly linking microbiota to hip fracture risk. Material and methods: Observational, analytical, case–control study. The sample consisted of 50 patients and it was distributed as follows: 25 elderly patients with fragility hip fracture and 25 subjects without fracture. The intestinal microbiota was determined by DNA extraction from stool samples and 16S ribosomal DNA sequencing after generation of gene libraries. Results: Alpha diversity revealed an elevation of the estimators for the taxonomic class level in the hip fracture group. The orders Bacteroidales, Oscillospirales, Lachnospirales, Peptostreptococcales-Tissierellales and Enterobacterales were the dominant orders in both groups. In patients with fracture, a significant percentage increase in the orders Bacteroidales (p<.001) and Peptostreptococcales-Tissierellales (p<.005) was observed, as well as a decrease in the orders Lachnospirales (p<.001) compared to controls. Conclusions: This study has found an association between a specific microbiota in elderly patients with fragility hip fracture. These findings open the door to new strategies to prevent hip fractures. Modification of the microbiota through probiotics may prove to be an effective method to reduce the risk of hip fracture.(AU)

[Translated article] Pilot study to determine the association between gut microbiota and fragility hip fracture.

INTRODUCTION: Hip fractures are the most common cause of hospital admission to orthopaedic departments in Europe and they generate a major health problem. Therefore, it is of great interest to identify additional risk factors that will help us to better understand the pathophysiology of these fractures and improve our preventive capacity. There is sufficient data to support the theory of modulation of bone mass by gut microbiota (osteomicrobiology); however, there is a lack of human clinical studies directly linking microbiota to hip fracture risk. MATERIAL AND METHODS: Observational, analytical, case-control study. The sample consisted of 50 patients and it was distributed as follows: 25 elderly patients with fragility hip fracture and 25 subjects without fracture. The intestinal microbiota was determined by DNA extraction from stool samples and 16S ribosomal DNA sequencing after generation of gene libraries. RESULTS: Alpha diversity revealed an elevation of the estimators for the taxonomic class level in the hip fracture group. The orders Bacteroidales, Oscillospirales, Lachnospirales, Peptostreptococcales-Tissierellales and Enterobacterales were the dominant orders in both groups. In patients with fracture, a significant percentage increase in the orders Bacteroidales (p<.001) and Peptostreptococcales-Tissierellales (p<.005) was observed, as well as a decrease in the orders Lachnospirales (p<.001) compared to controls. CONCLUSIONS: This study has found an association between a specific microbiota in elderly patients with fragility hip fracture. These findings open the door to new strategies to prevent hip fractures. Modification of the microbiota through probiotics may prove to be an effective method to reduce the risk of hip fracture.

Pilot study to determine the association between gut microbiota and fragility hip fracture. / Estudio piloto para determinar la asociación entre la microbiota intestinal y la fractura de cadera por fragilidad.

INTRODUCTION: Hip fractures are the most common cause of hospital admission to orthopaedic departments in Europe and they generate a major health problem. Therefore, it is of great interest to identify additional risk factors that will help us to better understand the pathophysiology of these fractures and improve our preventive capacity. There is sufficient data to support the theory of modulation of bone mass by gut microbiota (osteomicrobiology); however, there is a lack of human clinical studies directly linking microbiota to hip fracture risk. MATERIAL AND METHODS: Observational, analytical, case-control study. The sample consisted of 50 patients and it was distributed as follows: 25 elderly patients with fragility hip fracture and 25 subjects without fracture. The intestinal microbiota was determined by DNA extraction from stool samples and 16S ribosomal DNA sequencing after generation of gene libraries. RESULTS: Alpha diversity revealed an elevation of the estimators for the taxonomic class level in the hip fracture group. The orders Bacteroidales, Oscillospirales, Lachnospirales, Peptostreptococcales-Tissierellales and Enterobacterales were the dominant orders in both groups. In patients with fracture, a significant percentage increase in the orders Bacteroidales (p<.001) and Peptostreptococcales-Tissierellales (p<.005) was observed, as well as a decrease in the orders Lachnospirales (p<.001) compared to controls. CONCLUSIONS: This study has found an association between a specific microbiota in elderly patients with fragility hip fracture. These findings open the door to new strategies to prevent hip fractures. Modification of the microbiota through probiotics may prove to be an effective method to reduce the risk of hip fracture.

Two alkaline motifs in the Lactobacillus salivarius Lv72 OppA surface are important to its adhesin function.

Glycosaminoglycans are involved in the attachment of Lactobacillus salivarius Lv72, a strain of vaginal origin, to HeLa cell cultures, indicating that they play a fundamental role in the attachment of mutualistic bacteria to the epithelium lining cavities where the normal microbiota thrives. The bacterial OppA protein has been proposed as an adhesin involved in this adherence since, once purified, it significantly interferes with attachment of the lactobacilli to HeLa cell cultures. In this article, the role of OppA is confirmed through the determination of its location at the cell surface and its ability to promote Lactobacillus casei and Lactococcus lactis adherence to eukaryotic cell cultures upon cloning and expression of oppA in these bacteria. The OppA sequence showed five potential domains for glycosaminoglycan-binding, and structural modelling of the protein showed that two of them were located in the vicinity of an OppA superficial groove whose width approached the diameter of the helical form of heparin in solution. Their involvement in the binding was demonstrated through substitution of critical basic amino acids by acidic ones, which resulted in loss of affinity for heparan sulphate and chondroitin sulphate depending on the domain mutated, suggesting that there might be a certain degree of specialisation. In addition, circular dichroism analysis showed that the spectrum changes induced by OppA-heparan sulphate binding were attenuated by the variant proteins, indicating that these motifs are the OppA recognition domains for the eukaryotic cell surface.

Shifts in gut microbiota composition in an APP/PSS1 transgenic mouse model of Alzheimer's disease during lifespan.

Alzheimer's disease (AD) is the most common form of dementia and one of the major causes of disability and dependency in older people. Accumulating evidences link gut microbiota with different diseases and its relationship with neurodegenerative diseases is becoming most intriguing. This study was aimed to compare the gut microbiota of transgenic APP/PS1 (TG) mice, a well-established deterministic mouse model of AD, with their C57BL/6 wild-type (WT) littermates. Faecal samples were collected from 3-, 6- and 24-month-old mice and analysed by pyrosequencing of the V1-V3 region of the bacterial 16S rRNA genes. Bacterial profiles were similar in all young mice (3 months old), and started to diverge so that 6-month-old WT and TG mice had different and more diverse microbiota. During ageing, Turicibacteriaceae (typical mice bacterial group) and Rikenellaceae increased in all groups, although total Bacteroidetes remained stable. TG mice were characterized by an increase in Proteobacteria after 6 months, particularly the genus Sutterella (Betaproteobacteria), interestingly also increased in autism disorder. Also, the inflammation related family Erysipelotrichaceae was more abundant in TG mice at 24 months compared to wild-type control. In summary, AD pathology in mice shifts the gut microbiota towards profiles that share features with autism and inflammatory disorders. SIGNIFICANCE AND IMPACT OF THE STUDY: Alzheimer's disease is a neurodegenerative disease and neuroinflammation in the central nervous system appears to have a pivotal role. Using the transgenic APP/PS1 (TG) mouse model, we successfully characterized how AD pathology shifted gut microbiota composition during ageing towards an inflammation related bacterial profile related to Proteobacteria and Erysipelotrichaceae and suggest that these changes could contribute to disease progression and severity. Microbiota-targeted interventions could therefore represent a strategy to postpone disease symptoms.

Prenatal Ultrasound Diagnosis of a Cyst of the Oral Cavity: An Unusual Case of Thyroglossal Duct Cyst Located on the Tongue Base.

We describe a case of a lingual thyroglossal duct cyst diagnosed prenatally by ultrasound at 26 weeks of gestation. The follow-up ultrasound scans revealed no changes in the cyst measurement. Surgical treatment was performed without any complication 72 hours after delivery with good results.

Impact of Fishmeal Replacement in Diets for Gilthead Sea Bream (Sparus aurata) on the Gastrointestinal Microbiota Determined by Pyrosequencing the 16S rRNA Gene.

Recent studies have demonstrated the impact of diet on microbiota composition, but the essential need for the optimization of production rates and costs forces farms and aquaculture production to carry out continuous dietary tests. In order to understand the effect of total fishmeal replacement by vegetable-based feed in the sea bream (Sparus aurata), the microbial composition of the stomach, foregut, midgut and hindgut was analysed using high-throughput 16S rDNA sequencing, also considering parameters of growth, survival and nutrient utilisation indices.A total of 91,539 16S rRNA filtered-sequences were analysed, with an average number of 3661.56 taxonomically assigned, high-quality sequences per sample. The dominant phyla throughout the whole gastrointestinal tract were Actinobacteria, Protebacteria and Firmicutes. A lower diversity in the stomach in comparison to the other intestinal sections was observed. The microbial composition of the Recirculating Aquaculture System was totally different to that of the sea bream gastrointestinal tract. Total fishmeal replacement had an important impact on microbial profiles but not on diversity. Streptococcus (p-value: 0.043) and Photobacterium (p-value: 0.025) were highly represented in fish fed with fishmeal and vegetable-meal diets, respectively. In the stomach samples with the vegetable diet, reads of chloroplasts and mitochondria from vegetable dietary ingredients were rather abundant. Principal Coordinate Analysis showed a clear differentiation between diets in the microbiota present in the gut, supporting the presence of specific bacterial consortia associated with the diet.Although differences in growth and nutritive parameters were not observed, a negative effect of the vegetable diet on the survival rate was determined. Further studies are required to shed more light on the relationship between the immune system and sea bream gastrointestinal tract microbiota and should consider the modulation of the microbiota to improve the survival rate and nutritive efficacy when using plant-based diets.

Understanding gut microbiota in elderly's health will enable intervention through probiotics.

Today, advances in the public health system of most countries have managed to extend notably life expectancy, however, elderly's health remain as a very serious concern. The lifelong stimulation of innate and adaptive immune systems leads to immunosenescence and, as result, to a low ability to produce immunoglobulins against pathogens but also to a low-grade chronic inflammatory state (inflammaging) that is linked to most age-related health problems, such as dementia, Alzheimer or atherosclerosis. This inflammatory state could make the host more sensitive to intestinal microbes, or vice versa, as changes in the gut microbiota composition are related to the progression of diseases and frailty in the elderly population. It was considered that gut microbiota changed during aging, with an increase of Bacteroidetes vs. Firmicutes proportion and a reduction of bifidobacterial counts, however recent studies reported a great inter-individual variation among elderly and a significant relationship between gut microbiota, diet and institution or community living. Intervention studies of probiotics and prebiotics in elderly are not very abundant, but most cases showed that Bifidobacterium populations can efficiently be stimulated with a concomitant decrease of Enterobacteria. Furthermore, also some studies demonstrated that probiotics decreased the synthesis of pro-inflammatory cytokines which are upregulated in the elderly, such as interleukin (IL)-8, IL-6 or tumour necrosis factor ?, among others, and they increased the levels of activated lymphocytes, natural killer cells, phagocytic activity and even showed a greater response to influenza vaccination. This suggests that direct manipulation of the gut microbiota may improve adaptive immune response and reduce inflammatory secretions, therefore compensating immunosenescence effects, however, there are no records of their effect on clinical symptoms or risk for disease. Those facts reveal that this is an open research field with very good scientific perspectives and above all they could bring likely improvements in the wellbeing of our seniors.

¿Existe una relación entre la microbiota intestinal, el consumo de probióticos y la modulación del peso corporal? / Is there a relationship between gut microbiota, probiotics and body weight modulation?

Introducción: Estudios científicos recientes indican que la microbiota intestinal puede jugar un papel importante en la modulación del peso corporal del hospedador. Objetivo: En este artículo se presenta una revisión actualizada de la literatura científica sobre el papel potencial de la microbiota intestinal y del consumo de probióticos en el peso corporal del hospedador, incluyendo la predisposición y la prevención del sobrepeso y la obesidad. Resultados y conclusiones: El empleo de probióticos en diferentes etapas de crecimiento, tanto en el hospedador animal como el humano, está habitualmente asociado a beneficios en la salud. Es evidente que los beneficios asociados al crecimiento no implican necesariamente un aumento del tejido adiposo ni tampoco una predisposición al sobrepeso o la obesidad. Hasta el momento, los datos que asocian un tipo de microorganismos específicos con la obesidad humana no son concluyentes ya que no determinan si es dicha microbiota la que juega una función causativa de la obesidad (fenómeno primario), o si es la microbiota intestinal la que está modulada en respuesta a dietas obesogénicas u otros factores relacionados con la patogénesis de esta condición (fenómeno secundario). Los estudios dirigidos a la modulación de la microbiota intestinal para prevenir o controlar la obesidad del hospedador, incluido el uso de probióticos, muestran resultados prometedores. De hecho, el consumo de probióticos en el entorno materno-infantil podría contribuir al control del peso corporal en etapas posteriores mediante la modulación de la microbiota intestinal infantil. Sin embargo, son necesarios más estudios que empleen ensayos aleatorizados, doble ciego y controlados por placebo para poder demostrar la eficacia de cepas probióticas específicas para la prevención o el tratamiento del sobrepeso y la obesidad. En el marco de la actual pandemia de obesidad, el empleo de cepas probióticas de las que se disponga de evidencia científica sobre un efecto beneficioso frente a determinados factores de riesgo asociados a la obesidad podría servir, junto con cambios en la dieta y el fomento de la actividad física, a la modulación del peso corporal (AU)

Introduction: Recent scientific studies show that gut microbiota may play an important role in the modulation of the body weight of the host. Objective: The aim of this article is to present an updated review of the scientific literature dealing with the potential roles of the gut microbiota and probiotics on the body weight of the host, including the predisposition to and prevention of overweight and obesity. Results and conclusions: The use of probiotics in different growth stages, both in human and animal hosts, is usually associated to a beneficial effect to the host's health. Admittedly, benefits associated to growth do not necessarily imply an increase in the adipose tissue or a predisposition to overweight or obesity. At present, the data that link the presence of specific gut microbial groups with obesity are controversial since it is unknown if they represent a cause or a consequence of obesity-associated diets and/or any other factor related to the pathogenesis of this condition. Studies dealing with the modulation of the gut microbiota to prevent or control obesity in the host, including the use of probiotics, are promising. In fact, probiotic intake in the mother-infant context might contribute to the control of the adult body weight by modulating the infant gut microbiota. However, well-designed randomized double-blind placebo-controlled trials are required to demonstrate the efficacy of specific probiotic strains for prevention or treatment of overweight and obesity. In the frame of the current obesity pandemics, use of probiotic strains with scientifically-substantiated properties against obesity risk factors may constitute a future approach, complementary to changes in diet and life style, for the modulation of the body weight (AU)

Characterization of a fibronectin-binding protein from Lactobacillus casei BL23.

AIMS: To characterize the functionality of the Lactobacillus casei BL23 fbpA gene encoding a putative fibronectin-binding protein. METHODS AND RESULTS: Adhesion tests showed that L. casei BL23 binds immobilized and soluble fibronectin in a protease-sensitive manner. A mutant with inactivated fbpA showed a decrease in binding to immobilized fibronectin and a strong reduction in the surface hydrophobicity as reflected by microbial adhesion to solvents test. However, minor effects were seen on adhesion to the human Caco-2 or HT-29 cell lines. Purified 6X(His)FbpA bound to immobilized fibronectin in a dose-dependent manner. Western blot experiments with FbpA-specific antibodies showed that FbpA could be extracted from the cell surface by LiCl treatment and that protease digestion of the cells reduced the amount of extracted FbpA. Furthermore, surface exposition of FbpA was detected in other L. casei strains by LiCl extraction and whole-cell ELISA. CONCLUSIONS: FbpA can be found at the L. casei BL23 surface and participates in cell attachment to immobilized fibronectin. We showed that FbpA is an important, but not the only, factor contributing to fibronectin binding in BL23 strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report showing the involvement of FbpA in fibronectin binding in L. casei BL23 and represents a new contribution to the study of attachment factors in probiotic bacteria.

Induction of systemic and mucosal immune response and decrease in Streptococcus pneumoniae colonization by nasal inoculation of mice with recombinant lactic acid bacteria expressing pneumococcal surface antigen A

Mucosal epithelia constitute the first barriers to be overcome by pathogens during infection. The induction of protective IgA in this location is important for the prevention of infection and can be achieved through different mucosal immunization strategies. Lactic acid bacteria have been tested in the last few years as live vectors for the delivery of antigens at mucosal sites, with promising results. In this work, Streptococcus pneumoniae PsaA antigen was expressed in different species of lactic acid bacteria, such as Lactococcus lactis, Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus helveticus. After nasal inoculation of C57Bl/6 mice, their ability to induce both systemic (IgG in serum) and mucosal (IgA in saliva, nasal and bronchial washes) anti-PsaA antibodies was determined. Immunization with L. lactis MG1363 induced very low levels of IgA and IgG, possibly by the low amount of PsaA expressed in this strain and its short persistence in the nasal mucosa. All three lactobacilli persisted in the nasal mucosa for 3 days and produced a similar amount of PsaA protein (150-250 ng per 109 CFU). However, L. plantarum NCDO1193 and L. helveticus ATCC15009 elicited the highest antibody response (IgA and IgG). Vaccination with recombinant lactobacilli but not with recombinant L. lactis led to a decrease in S. pneumoniae recovery from nasal mucosa upon a colonization challenge. Our results confirm that certain Lactobacillus strains have intrinsic properties that make them suitable candidates for mucosal vaccination experiments.

Characterization of faecal enterococci from rabbits for the selection of probiotic strains.

AIMS: To characterize the facultative anaerobic intestinal microbiota of healthy rabbits, especially enterococci, for the selection of potential probiotic strains. METHODS AND RESULTS: Phenotypic and molecular methods were used to identify enterococcal isolates. Results obtained indicated that enterococcal microbiota widely varied among individuals both in size and in composition. Antibacterial and haemolytic activities, and resistance to acid and bile salts were determined. A small group of strains produced bacteriocins active against listeriae and indigenous clostridia and therefore they were selected as potential probiotics. One such strain, 8G, was assayed for colonization capacity. Results obtained suggested that the fate of the introduced strain depended on the composition of the enterococcal indigenous microbiota. CONCLUSIONS: Enterococcus faecalis and Ent. faecium are the predominant enterococcal species in the gut of rabbits. Other species of lactic acid bacteria were not recovered. SIGNIFICANCE AND IMPACT OF THE STUDY: The enterococcal intestinal microbiota of healthy rabbits has been characterized in detail. Monitoring the fate of an introduced probiotic in vivo is required in order to evaluate potential probiotic strains.

Expression of Streptococcus pneumoniae antigens, PsaA (pneumococcal surface antigen A) and PspA (pneumococcal surface protein A) by Lactobacillus casei

number of recent research works in lactic acid bacteria aim towards the design of new strains that could be used as live vectors for the delivery of antigens for oral vaccination, or other therapeutic molecules. In this work, an inducible expression system based on the Lactobacillus casei lactose operon promoter was used to express three important surface antigens of Streptococcus pneumoniae in this lactic acid bacterium: a virulence-related pneumococcal surface antigen (PsaA) and two variants of the virulence factor PspA (pneumococcal surface protein A). Expression of the three proteins was induced upon growth on lactose and strongly repressed by glucose. These proteins were produced intracellularly. Also, secretion to the growth medium was achieved by means of a fusion to the secreting and processing signals from the L. casei surface proteinase. Interestingly, while secreted PspA proteins were found in the culture supernatants, PsaA remained trapped in the cell wall. Expression of pneumococcal antigens in a food-grade organism opens an alternative for mucosal vaccination against this important pathogen. © 2003 Federation of European Microbiological Societies.

Construction of compatible wide-host-range shuttle vectors for lactic acid bacteria and Escherichia coli.

A new collection of shuttle cloning vectors has been constructed that can be used in a broad host range, because they carry replication origins which are functional in Escherichia coli (p15A, pWV01, ColE1), Lactococcus lactis, lactobacilli, and Bacillus subtilis (pAMbeta1, pWV01). These plasmids contain the lacZ-T1T2 cassette from pJDC9, which allows the X-gal selection and cloning of DNA fragments that could cause plasmid instability in E. coli. In addition, they have been proved to be structurally and segregationally stable in Lactobacillus casei, in which their copy number has been determined by real-time quantitative PCR. Furthermore, the antibiotic resistance markers (beta-lactamase, chloramphenicol acetyl transferase, and erythromycin transacetylase) and the theta and rolling circle replicating origins have been combined to obtain this set of compatible plasmids (pIA family) that can be cotransformed, both in lactic acid bacteria and in E. coli.