Molecular detection of hemotrophic mycoplasmas among domiciled and free-roaming cats in Campo Grande, state of Mato Grosso do Sul, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Hemoplasmas in wild canids and felids in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Adição de óleo de Citrus sinensis na dieta de tilápia-do-Nilo: desempenho produtivo, perfil hematológico e atividade respiratória de leucócitos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Classe mollicutes na boca de pacientes com dependência química

Drug addiction is one of the biggest public health problems worldwide, not only by the dimensions of the problem, but also by the severity of the damage, creating favorable conditions for opportunistic microorganisms such as class Mollicutes. This study aims to evaluate the presence of the main species and genera of this group in the subgingival biofilm of drug addiction patients, comparing them with non-dependent subjects. For this purpose, data on systemic health conditions, socioeconomic characteristics, drug addiction from 72 patients with drug addiction kept in rehab clinics and 100 non-dependent patients who formed the control group were obtained. Intra and extraoral clinical examinations were performed and samples of subgingival plaque were collected through sterile absorbent paper cones. The presence of different genera and species of the class Mollicutes was evaluated by PCR using the specific primers and conditions for each microorganism. The statistical analysis was performed using the Chi-square test for comparisons of three or more variables and the Mann-Whitney test, with significance level of 5%. Out of species and genera evaluated, Mycoplasma salivarium showed correlation with gingival inflammation in both patient groups and was more frequently detected among drug addiction patients

Toll-like receptors 2, 3 and 4 and thymic stromal lymphopoietin expression in fatal asthma

Background Airway inflammation in asthma involves innate immune responses. Toll-like receptors (TLRs) and thymic stromal lymphopoietin (TSLP) are thought to be involved in airway inflammation, but their expression in asthmatics both large and small airways has not been investigated. Objective To analyse the expression of TLR2, TLR3, TLR4 and TSLP in large and small airways of asthmatics and compare their expression in smoking and non-smoking asthmatics; to investigate whether TLR expression is associated with eosinophilic or neutrophilic airway inflammation and with Mycoplasma pneumoniae and Chlamydophila pneumoniae infection. Methods Using immunohistochemistry and image analysis, we investigated TLR2, TLR3, TLR4 and TSLP expression in large and small airways of 24 victims of fatal asthma, FA, (13 non-smokers, 11 smokers) and nine deceased control subjects (DCtrl). TLRs were also measured in 18 mild asthmatics (MA) and 12 healthy controls (HCtrl). M. pneumoniae and C. pneumoniae in autopsy lung tissue were analysed using real-time polymerase chain reaction. Airway eosinophils and neutrophils were measured in all subjects. Results Fatal asthma patients had higher TLR2 in the epithelial and outer layers of large and small airways compared with DCtrls. Smoking asthmatics had lower TLR2 levels in the inner and outer layers of the small airways than non-smoking asthmatics. TSLP was increased in the epithelial and outer layers of the large airways of FA. FA patients had greater TLR3 expression in the outer layer of large airways and greater TLR4 expression in the outer layer of small airways. Eosinophilic airway inflammation was associated with TLR expression in the epithelium of FA. No bacterial DNA was detected in FA or DCtrls. MA and HCtrls had only a small difference in TLR3 expression. Conclusions and Clinical Relevance Increased expression of TLR 2, 3 and 4 and TSLP in fatal asthma may contribute to the acute inflammation surrounding asthma deaths.

Community-acquired pneumonia in Chile: the clinical relevance in the detection of viruses and atypical bacteria

Background Adult community-acquired pneumonia (CAP) is a relevant worldwide cause of morbidity and mortality, however the aetiology often remains uncertain and the therapy is empirical. We applied conventional and molecular diagnostics to identify viruses and atypical bacteria associated with CAP in Chile. Methods We used sputum and blood cultures, IgG/IgM serology and molecular diagnostic techniques (PCR, reverse transcriptase PCR) for detection of classical and atypical bacteria (Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumoniae) and respiratory viruses (adenovirus, respiratory syncytial virus (RSV), human metapneumovirus, influenza virus, parainfluenzavirus, rhinovirus, coronavirus) in adults >18 years old presenting with CAP in Santiago from February 2005 to September 2007. Severity was qualified at admission by Fine's pneumonia severity index. Results Overall detection in 356 enrolled adults were 92 (26%) cases of a single bacterial pathogen, 80 (22%) cases of a single viral pathogen, 60 (17%) cases with mixed bacterial and viral infection and 124 (35%) cases with no identified pathogen. Streptococcus pneumoniae and RSV were the most common bacterial and viral pathogens identified. Infectious agent detection by PCR provided greater sensitivity than conventional techniques. To our surprise, no relationship was observed between clinical severity and sole or coinfections. Conclusions The use of molecular diagnostics expanded the detection of viruses and atypical bacteria in adults with CAP, as unique or coinfections. Clinical severity and outcome were independent of the aetiological agents detected.

Análisis de la región intergénica ARNr 16-23S y del gen ARNr 23S en cepas de campo de Mycroplasma hyopneumoniae : estudio filogenético de los micoplasmas porcinos en base al gen ARNr 23S

Programa de doctorado: Sanidad animal

MetaVaccinology: a new vaccine discovery tool

In the last decade, the reverse vaccinology approach shifted the paradigm of vaccine discovery from conventional culture-based methods to high-throughput genome-based approaches for the development of recombinant protein-based vaccines against pathogenic bacteria. Besides reaching its main goal of identifying new vaccine candidates, this new procedure produced also a huge amount of molecular knowledge related to them. In the present work, we explored this knowledge in a species-independent way and we performed a systematic in silico molecular analysis of more than 100 protective antigens, looking at their sequence similarity, domain composition and protein architecture in order to identify possible common molecular features. This meta-analysis revealed that, beside a low sequence similarity, most of the known bacterial protective antigens shared structural/functional Pfam domains as well as specific protein architectures. Based on this, we formulated the hypothesis that the occurrence of these molecular signatures can be predictive of possible protective properties of other proteins in different bacterial species. We tested this hypothesis in Streptococcus agalactiae and identified four new protective antigens. Moreover, in order to provide a second proof of the concept for our approach, we used Staphyloccus aureus as a second pathogen and identified five new protective antigens. This new knowledge-driven selection process, named MetaVaccinology, represents the first in silico vaccine discovery tool based on conserved and predictive molecular and structural features of bacterial protective antigens and not dependent upon the prediction of their sub-cellular localization.

Therapeutic strategies for modulation of the vaginal microbiota

The vaginal microbiota of healthy women consists of a wide variety of anaerobic and aerobic bacteria, dominated by the genus Lactobacillus. The activity of lactobacilli is essential to protect women from genital infections and to maintain the natural healthy balance of the vaginal ecosystem. This role is particularly important during pregnancy because vaginal infection is one of the most important mechanisms for preterm birth. The most common vaginal disorder is bacterial vaginosis (BV). BV is a polymicrobial disorder, characterized by a depletion of lactobacilli and an increase in the concentration of other bacteria, including Gardnerella vaginalis, anaerobic Gram-negative rods, anaerobic Gram-positive cocci, Mycoplasma hominis, and Mobiluncus spp. An integrated molecular approach based on real-time PCR and PCR-DGGE was used to investigate the effects of two different therapeutic approaches on the vaginal microbiota composition. (i) The impact of a dietary supplementation with the probiotic VSL#3, a mixture of Lactobacillus, Bifidobacterium and Streptococcus strains, on the vaginal microbial ecology and immunological profiles of healthy women during late pregnancy was investigated. The intake was associated to a slight modulation of the vaginal microbiota and cytokine secretion, with potential implications in preventing preterm birth. (ii) The efficacy of different doses of the antibiotic rifaximin (100 mg/day for 5 days, 25 mg/day for 5 days, 100 mg/day for 2 days) on the vaginal microbiota of patients with BV enrolled in a multicentre, double-blind, randomised, placebo-controlled study was also evaluated. The molecular analyses demonstrated the ability of rifaximin 25 mg/day for 5 days to induce an increase of lactobacilli and a decrease of the BV-associated bacteria after antibiotic treatment, and a reduction of the complexity of the vaginal microbial communities. Thus, confirming clinical results, it represents the most effective treatment to be used in future pivotal studies for the treatment of BV.

Polymerizing activity and regulation of group B Streptococcus pilus 2a sortase C1

Group B Streptococcus [GBS; Streptococcus agalactiae] is the leading cause of life-threatening diseases in newborn and is also becoming a common cause of invasive diseases in non-pregnant, elderly and immune-compromised adults. Pili, long filamentous fibers protruding from the bacterial surface, have been discovered in GBS, as important virulence factors and vaccine candidates. Gram-positive bacteria build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates. Despite the availability of several crystal structures, pilus-related C sortases remain poorly characterized to date and their mechanisms of transpeptidation and regulation need to be further investigated. The available three-dimensional structures of these enzymes reveal a typical sortase fold except for the presence of a unique feature represented by an N-terminal highly flexible loop, known as the “lid”. This region interacts with the residues composing the catalytic triad and covers the active site, thus maintaining the enzyme in an auto-inhibited state and preventing the accessibility to the substrate. It is believed that enzyme activation may occur only after lid displacement from the catalytic domain. In this work we provide the first direct evidence of the regulatory role of the lid, demonstrating that it is possible to obtain in vitro an efficient polymerization of pilin subunits using an active C sortase lid mutant carrying a single residue mutation in the lid region. Moreover, biochemical analyses of this recombinant mutant reveal that the lid confers thermodynamic and proteolytic stability to the enzyme. A further characterization of this sortase active mutant showed promiscuity in the substrate recognition, as it is able to polymerize different LPXTG-proteins in vitro.

The role of bifidobacteria in newborn health and the intestinal microbial balance

Gut microbial acquisition during the early stage of life is an extremely important event since it affects the health status of the host. In this contest the healthy properties of the genus Bifidobacterium have a central function in newborns. The aim of this thesis was to explore the dynamics of the gut microbial colonization in newborns and to suggest possible strategies to maintain or restore a correct balance of gut bacterial population in infants. The first step of this work was to review the most recent studies on the use of probiotics and prebiotics in infants. Secondly, in order to prevent or treat intestinal disorders that may affect newborns, the capability of selected Bifidobacterium strains to reduce the amount of Enterobacteriaceae and against the infant pathogen Streptococcus agalactiae was evaluated in vitro. Furthermore, the ability of several commercial fibers to stimulate selectively the growth of bifidobacterial strains was checked. Finally, the gut microbial composition in the early stage of life in response to the intrapartum antibiotic prophylaxis (IAP) against group B Streptococcus was studied using q-PCR, DGGE and next generation sequencing. The results globally showed that Bifidobacterium breve B632 strain is the best candidate for the use in a synbiotic product coupled to a mixture of two selected prebiotic fibers (galactooligosaccharides and fructooligosaccharides) for gastrointestinal disorders in infants. Moreover, the early gut microbial composition was affected by IAP treatment with infants showing lower counts of Bifidobacterium spp. and Bacteroides spp. coupled to a decrement of biodiversity of bacteria, compared to control infants. These studies have shown that IAP could affect the early intestinal balance in infants and they have paved the way to the definition of new strategies alternative to antibiotic treatment to control GBS infection in pregnant women.

Repression of common bull sperm flora and in vitro impairment of sperm motility with Pseudomonas aeruginosa introduced by contaminated lubricant

Semen collected from clinically healthy bulls at an artificial insemination centre was examined for bacterial diversity. While bacteria that are normally present in the common flora of bovine semen were absent, such as Mycoplasma sp., Proteus sp. and Corynebacterium sp., all semen samples contained an unusually high number of Pseudomonas aeruginosa strains. Analysis via pulsed field gel electrophoresis demonstrated that one particular P. aeruginosa strain, present in a sealed bottle of lubricant, was widespread in bull semen. This strain was shown to secrete substances that inhibited both the growth of bacteria constituting the normal bull sperm flora and the motility of spermatozoa in vitro. This study demonstrated that commercially available lubricants might contain bacteria that can spread amongst breeding bulls and affect the quality of semen. Bacteriological controls and species' identification are necessary at several production levels, including lubricants and extenders, to ensure high semen quality and avoid the spread of pathogens.

Cross-sectional study of Streptococcus species in quarter milk samples of dairy cows in the canton of Bern, Switzerland

A total of 2538 quarter milk samples from 638 lactating dairy cows from 47 farms in the canton of Bern, Switzerland, were investigated for streptococci. A novel, simple and inexpensive laboratory method was used for the differentiation of Streptococcus species, and a risk factor analysis was carried out. The prevalence in the quarter milk samples was 0.2 per cent for Streptococcus agalactiae, 1.3 per cent for Streptococcus uberis, 1.3 per cent for Streptococcus dysgalactiae, 0.1 per cent for Enterococcus species and 2.9 per cent for minor Streptococcus species (designated Streptococcus-Lactococcus-Enterococcus [SLE] group). Based on the somatic cell count (SCC), S uberis and S dysgalactiae were classified as 'major' pathogens and the bacteria in the SLE group as 'minor' pathogens. For S uberis, S dysgalactiae and bacteria in the SLE group, the most significant risk factor was an intramammary infection (IMI) of a neighbouring quarter by the same pathogen. Other significant risk factors for S uberis infection were a positive California Mastitis Test (CMT) result and a SCC of more than 100,000 cells/ml. Significant risk factors for IMI with S dysgalactiae were a positive CMT result, teat injury and palpable abnormalities in the udder. Infection with bacteria in the SLE group was significantly associated with a SCC of more than 100,000 cells/ml, a lactation number of more than 2, the right rear quarter (as the location of infection) and a positive CMT result.

Intramammary infections with the contagious Staphylococcus aureus genotype B in Swiss dairy cows are associated with low prevalence of coagulase-negative staphylococci and Streptococcus spp

The association between the contagious Staphylococcus aureus genotype B (GTB) and the presence of coagulase-negative staphylococci (CNS) and Streptococcus spp. (non-agalactiae streptococci), was investigated, and the identification of problem herds without genotyping was evaluated. Milk samples from 10 herds with Staph. aureus GTB herd problems (PH cases) were compared with samples from 19 herds with at least one Staph. aureus isolate of non-B genotype (CH cases). All samples were bacteriologically analysed and Staph. aureus genotyping carried out using a ribosomal spacer-PCR. Cow and quarter prevalences of Staph. aureus, CNS and Streptococcus spp. differed significantly between PH and CH groups. PH cases were highly associated with decreased cow prevalences of CNS and Streptococcus spp. These altered prevalences also contributed significantly to the identification of problem herds without resorting to genotyping. Common herd-level risk factors did not explain the difference between the prevalences in PH and CH cases.

Streptococcus spp. and related bacteria: Their identification and their pathogenic potential for chronic mastitis - A molecular approach

Streptococcus spp. and related bacteria form a large group of organisms which are associated with bovine intramammary Infections (IMI). Some of them are the well-known mastitis pathogens Streptococcus uberis and Streptococcus agalactiae. In addition, there are a considerable number of these gram-positive, catalase-negative cocci (PNC) with unclear mastitic pathogenicity such as Aerococcus viridans which make the conventional diagnostics of PNC difficult. One diagnostic, API 20 Strep (API, Biomerieux) is recommended which, as a phenotypic assay, involves a series of miniaturized biochemical tests. Recently, preference is given to genotypic identification methods. In particular, sequencing of the 16S rRNA gene allows highly reproducible and accurate identification of bacteria and permits discovery of novel, clinically relevant bacteria. As a consequence, the aim of the present study was to compare identification of IMI-associated PNC by the API method as well as by sequencing of their 16S rRNA gene (16S). Furthermore, the correlation of these bacteria to bovine chronic mastitis and their phylogeny was investigated. 102 PNC isolated from single quarter milk samples were identified by API and 16S sequencing. Considering Streptococcus uberis, Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus agalactiae, both methods generated fully concordant results. In contrast, a very high disconcordance was observed for most of the other PNC, in particular Enterococcus spp., Aerococcus viridans and the viridans streptococci were shown as apathogenic. Lactococcus garvieae was found to be an opportunistic pathogen causing IMI during late lactation. In addition, PNC isolated from milk were frequently observed together with other bacteria, in particular with Staphylococcus spp. In these cases, the levels of somatic cell counts (SCC) were determined by the specific PNC present in the sample. Considering PNC phylogeny based on 16S sequencing, 3 major clusters were observed. They included all the common mastitis pathogens (cluster I), the Lactococcus spp., Enterococcus spp. and Aerococcus spp. (cluster II) and all the viridans streptococci (cluster III).