Antibiosis and dark-pigments secretion by the phytopathogenic and environmental fungal species after interaction in vitro with a Bacillus subtilis isolate

In this work, different reactions in vitro between an environmental bacterial isolate and fungal species were related. The Gram-positive bacteria had terminal and subterminal endospores, presented metabolic characteristics of mesophilic and acidophilic growth, halotolerance, positive to nitrate reduction and enzyme production, as caseinase and catalase. The analysis of partial sequences containing 400 to 700 bases of the 16S ribosomal RNA gene showed identity with the genus Bacillus. However, its identity as B. subtilis was confirmed after analyses of the rpoB, gyrA, and 16S rRNA near-full-length sequences. Strong inhibitory activity of environmental microorganisms, such as Penicillium sp, Aspergillus flavus, A. niger, and phytopathogens, such as Colletotrichum sp, Alternaria alternata, Fusarium solani and F. oxysporum f.sp vasinfectum, was shown on co-cultures with B. subtilis strain, particularly on Sabouraud dextrose agar (SDA) and DNase media. Red and red-ochre color pigments, probably phaeomelanins, were secreted by A. alternata and A. niger respectively after seven days of co-culture.

Complete Genome Sequence of Mycoplasma suis and Insights into Its Biology and Adaption to an Erythrocyte Niche

Mycoplasma suis, the causative agent of porcine infectious anemia, has never been cultured in vitro and mechanisms by which it causes disease are poorly understood. Thus, the objective herein was to use whole genome sequencing and analysis of M. suis to define pathogenicity mechanisms and biochemical pathways. M. suis was harvested from the blood of an experimentally infected pig. Following DNA extraction and construction of a paired end library, whole-genome sequencing was performed using GS-FLX (454) and Titanium chemistry. Reads on paired-end constructs were assembled using GS De Novo Assembler and gaps closed by primer walking; assembly was validated by PFGE. Glimmer and Manatee Annotation Engine were used to predict and annotate protein-coding sequences (CDS). The M. suis genome consists of a single, 742,431 bp chromosome with low G+C content of 31.1%. A total of 844 CDS, 3 single copies, unlinked rRNA genes and 32 tRNAs were identified. Gene homologies and GC skew graph show that M. suis has a typical Mollicutes oriC. The predicted metabolic pathway is concise, showing evidence of adaptation to blood environment. M. suis is a glycolytic species, obtaining energy through sugars fermentation and ATP-synthase. The pentose-phosphate pathway, metabolism of cofactors and vitamins, pyruvate dehydrogenase and NAD(+) kinase are missing. Thus, ribose, NADH, NADPH and coenzyme A are possibly essential for its growth. M. suis can generate purines from hypoxanthine, which is secreted by RBCs, and cytidine nucleotides from uracil. Toxins orthologs were not identified. We suggest that M. suis may cause disease by scavenging and competing for host nutrients, leading to decreased life-span of RBCs. In summary, genome analysis shows that M. suis is dependent on host cell metabolism and this characteristic is likely to be linked to its pathogenicity. The prediction of essential nutrients will aid the development of in vitro cultivation systems.

Anaerobic degradation of linear alkylbenzene sulfonate (LAS) in fluidized bed reactor by microbial consortia in different support materials

Four anaerobic fluidized bed reactors filled with activated carbon (R1), expanded clay (R2), glass beads (R3) and sand (R4) were tested for anaerobic degradation of LAS. All reactors were inoculated with sludge from a UASB reactor treating swine wastewater and were fed with a synthetic substrate supplemented with approximately 20 mg l(-1) of LAS, on average. To 560 mg l(-1) COD influent, the maximum COD and LAS removal efficiencies were mean values of 97 +/- 2% and 99 +/- 2%, respectively, to all reactors demonstrating the potential applicability of this reactor configuration for treating LAS. The reactors were kept at 30 degrees C and operated with a hydraulic retention time (HRT) of 18 h. The use of glass beads and sand appear attractive because they favor the development of biofilms capable of supporting LAS degradation. Subsequent 16S rRNA gene sequencing and phylogenetic analysis of samples from reactors R3 and R4 revealed that these reactors gave rise to broad microbial diversity, with microorganisms belonging to the phyla Bacteroidetes, Firmicutes, Actinobacteria and Proteobacteria, indicating the role of microbial consortia in degrading the surfactant LAS. (C) 2010 Elsevier Ltd. All rights reserved.

Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells

The objective of the present study was to characterize the innate immune responses induced by in vitro stimulation of bovine primary mammary epithelial cells (bMEC) using gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. Quantitative real-time PCR (qRT-PCR) was employed to examine the mRNA expression of a panel of 22 cytokines, chemokines, beta-defensins and components of the Toll-Like Receptor signaling pathway. Stimulation of bMEC with LPS for 24 h elicited a marked increase in mRNA expression for IL-1 beta, IL-8, TNF alpha, CXCL6 and beta-defensin while members of the Toll-Like Receptor pathway.. although present, were largely unaffected. Surprisingly, stimulation of these cells with LTA for 24 h did not significantly alter the expression of these genes. A time course of the expression of IL-1 beta, IL-8, TNF alpha, CXCL6 and beta-defensin was subsequently performed. The mRNA levels of all genes increased rapidly after stimulation for 2-4 h with both LPS and LTA but only the former treatment resulted in sustained responses. In contrast, the increased gene expression for LTA stimulated cells returned to resting levels after 8-16 h with the exception of beta-defensin, which remained up-regulated. The limited and unsustained cytokine response to LTA may explain why mastitis caused by gram-positive bacteria has greater potential for chronic intra-mammary infection than gram-negative infection. It was concluded that bovine mammary epithelial cells have a strong but differential capacity to mount innate immune responses to bacterial cell wall components. Crown Copyright (c) 2005 Published by Elsevier Ltd. All rights reserved.

Rheumatic Fever and Rheumatic Heart Disease: Cellular Mechanisms Leading Autoimmune Reactivity and Disease

Rheumatic fever (RF) is an autoimmune disease caused by the gram-positive bacteria Streptococcus pyogenes that follows a nontreated throat infection in susceptible children. The disease manifests as polyarthritis, carditis, chorea, erythema marginatum, and/or subcutaneous nodules. Carditis, the most serious complication, occurs in 30% to 45% of RF patients and leads to chronic rheumatic heart disease (RHD), which is characterized by progressive and permanent valvular lesions. In this review, we will focus on the genes that confer susceptibility for developing the disease, as well as the innate and adaptive immune responses against S. pyogenes during the acute rheumatic fever episode that leads to RHD autoimmune reactions. The disease is genetically determined, and some human leukocyte antigen class II alleles are involved with susceptibility. Other single nucleotide polymorphisms for TNF-alpha and mannan-binding lectin genes were reported as associated with RF/RHD. T cells play an important role in RHD heart lesions. Several autoantigens were already identified, including cardiac myosin epitopes, vimentin, and other intracellular proteins. In the heart tissue, antigen-driven oligoclonal T cell expansions were probably the effectors of the rheumatic heart lesions. These cells are CD4(+) and produced inflammatory cytokines (TNF alpha and IFN gamma). Molecular mimicry is the mechanism that mediated the cross-reactions between streptococcal antigens and human proteins. The elucidation of chemokines and their receptors involved with the recruitment of Th1, Th2, and Th17 cells, as well as the function of T regulatory cells in situ will certainly contribute to the delineation of the real picture of the heart lesion process that leads to RHD.

The efficacy of functional endoscopic sinus surgery in the evolution of fever of unknown origin in ICU patients

Conclusion: Functional endoscopic sinus surgery (FESS) was found to be effective in treating fever of unknown origin (FUO) in intensive care unit (ICU) patients with rhinosinusitis, with 62% of patients showing improvement within 5 days of the procedure. Objective: To correlate improvement in FUO with FESS drainage of the paranasal sinuses. Methods: Fifty patients that developed FUO during ICU stay, with CT findings suggestive of rhinosinusitis, and showed no improvement in fever after clinical treatment underwent FESS for drainage of the paranasal sinuses and were evaluated for postoperative improvement of fever. Results: The study sample consisted of 50 patients (74% of whom were male, mean age 48.1 years). The most frequent diagnoses at ICU admission were tetanus, pulmonary disease, and cardiovascular disease. In all, 68% of patients underwent nasogastric or enteral intubation. CT scanning most commonly showed involvement of the sphenoid sinus. In 54% of cases, sinusitis was bilateral and extended throughout the maxillary, ethmoidal, and sphenoidal sinuses. Sinusectomy was performed in all patients, and pathological secretion in the paranasal sinuses was seen in 52% of patients during surgery. Gram-negative bacteria were the most commonly isolated organisms, followed by Gram-positive bacteria and fungi. Improvement of fever was found in 82% of patients after FESS; 38% of these improved within the first 48 h post-procedure, and the remaining 62% within the first 5 postoperative days.

The ultrastructure of Macropodinium moiri and revised diagnosis of the Macropodiniidae (Litostomatea : Trichostomatia)

The ultrastructural features of Macropodinium moiri were investigated. The somatic cortex is composed of two lateral non-ciliated zones covered with trapezoidal plates and separated by a trough-like dorsoventral groove (DVG) which divides the cell into left and right halves. The somatic kineties occupy the margins of the DVG and are composed of monokinetids whose infraciliature shows a typical litostome pattern. The pellicular plates are lamellate, and separated by V-shaped grooves which are lined by thick-walled vacuoles. The DVG cortex is composed of electron-opaque U-shaped ribs which alternate with electron-lucent saccular structures. The DVG surface is composed of small regular pellicular sacs built up to form the ridges of the dorsal DVG. The vestibulum forms a laterally compressed cone with left/right differentiation. The basal section of its non-ciliated right side is internally lined (outer to innermost) by longitudinal fibres, nematodesmata and transverse microtubular ribbons. The left side bears the vestibular kineties and in its basal section is lined (outer to innermost) by small nematodesmata and transverse tubules. Cytoplasmic organelles include endoplasmic reticulum, starch granules and a single contactile vacuole surrounded by patches of nephridioplasm. Hydrogenosomes are absent and coccoid Gram-positive bacteria lie under the ciliated portions of the cell. This set of characteristics differs significantly from those of the all other trichostomes; Macropodiniidae is therefore designated Trichostomatia incertae sedis. A revised familial diagnosis of the Macropodiniidae is proposed.

Antimicrobial and cytotoxic assessment of marine cyanobacteria - synechocystis and synechococcus

Aqueous extracts and organic solvent extracts of isolated marine cyanobacteria strains were tested for antimicrobial activity against a fungus, Gram-positive and Gram-negative bacteria and for cytotoxic activity against primary rat hepatocytes and HL-60 cells. Antimicrobial activity was based on the agar diffusion assay. Cytotoxic activity was measured by apoptotic cell death scored by cell surface evaluation and nuclear morphology. A high percentage of apoptotic cells were observed for HL-60 cells when treated with cyanobacterial organic extracts. Slight apoptotic effects were observed in primary rat hepatocytes when exposed to aqueous cyanobacterial extracts. Nine cyanobacteria strains were found to have antibiotic activity against two Gram-positive bacteria, Clavibacter michiganensis subsp. insidiosum and Cellulomonas uda. No inhibitory effects were found against the fungus Candida albicans and Gram-negative bacteria. Marine Synechocystis and Synechococcus extracts induce apoptosis in eukaryotic cells and cause inhibition of Gram-positive bacteria. The different activity in different extracts suggests different compounds with different polarities.

Bacterial inter-species communication mediated by the autoinducer-2 signal

Dissertation presented to obtain the Ph.D degree in Biology by Universidade Nova de Lisboa, Instituto de Tecnologia Química e Biológica, Instituto Gulbenkian de Ciência.

Infections After Liver Transplantation: A Retrospective, Single-Center Study

Objective. To access the incidence of infectious problems after liver transplantation (LT). Design. A retrospective, single-center study. Materials and Methods. Patients undergoing LT from January 2008 to December 2011 were considered. Exclusion criterion was death occurring in the first 48 hours after LT. We determined the site of infection and the bacterial isolates and collected and compared recipient’s variables, graft variables, surgical data, post-LT clinical data. Results. Of the 492 patients who underwent LT and the 463 considered for this study, 190 (Group 1, 41%) developed at least 1 infection, with 298 infections detected. Of these, 189 microorganisms were isolated, 81 (51%) gram-positive bacteria (most frequently Staphylococcus spp). Biliary infections were more frequent (mean time of 160.4 167.7 days after LT); from 3 months after LT, gram-negative bacteria were observed (57%). Patients with infections after LT presented lower aminotransferase levels, but higher requirements in blood transfusions, intraoperative vasopressors, hemodialysis, and hospital stay. Operative and cold ischemia times were similar. Conclusion. We found a 41% incidence of all infections in a 2-year follow-up after LT. Gram-positive bacteria were more frequent isolated; however, negative bacteria were commonly isolated later. Clinical data after LT were more relevant for the development of infections. Donors’ variables should be considered in future analyses.

Regulation and function of the cell wall polymer lipoteichoic acid in staphylococcus aureus

A thesis submitted for the Degree of Master in Medical microbiology

Comparative study of survivor and nonsurvivor sepsis patients in a university hospital

To determine parameters associated with the evolution of sepsis, a five-year retrospective study was conducted in a university hospital. One hundred and four consecutive sepsis patients were evaluated, of whom 55.8% were men. The mortality was 68.3% and was associated with older age (p<0.05). Chronic comorbidities and infection site were not associated with prognosis. Gram-positive bacteria were more frequently identified in survivors (p<0.05), while non-detection of the germ was associated with mortality (p<0.01). Appropriate use of antibiotics (germ sensitive to at least one drug administered) was associated with survival (p<0.0001) while inappropriate use (p<0.05) or empirical use (p<0.01) were more frequent in nonsurvivors. Leukocytosis was the main abnormality (54.8%) detected on diagnosis, from the leukocyte count. During the evolution, normal leukocyte count was associated with survival (p<0.01) and leukocytosis with mortality (p<0.05). In conclusion, mortality was associated with nondetection of the pathogen, leukocytosis during the evolution of the sepsis and inappropriate or empirical use of antimicrobials. Evidence-based treatment that is directed towards modifiable risk factors might improve the prognosis for sepsis patients.

Aerobic bacterial profile and antibiotic resistance in patients with diabetic foot infections

ABSTRACTINTRODUCTION: This study aimed to determine the frequencies of bacterial isolates cultured from diabetic foot infections and assess their resistance and susceptibility to commonly used antibiotics.METHODS: This prospective study included 41 patients with diabetic foot lesions. Bacteria were isolated from foot lesions, and their antibiotic susceptibility pattern was determined using the Kirby-Bauer disk diffusion method and/or broth method [minimum inhibitory concentration (MIC)].RESULTS: The most common location of ulceration was the toe (54%), followed by the plantar surface (27%) and dorsal portion (19%). A total of 89 bacterial isolates were obtained from 30 patients. The infections were predominantly due to Gram-positive bacteria and polymicrobial bacteremia. The most commonly isolated Gram-positive bacteria were Staphylococcus aureus, followed by Staphylococcus saprophyticus, Staphylococcus epidermidis, Streptococcus agalactiae, and Streptococcus pneumoniae. The most commonly isolated Gram-negative bacteria were Proteus spp. and Enterobacterspp., followed by Escherichia coli, Pseudomonasspp., and Citrobacterspp. Nine cases of methicillin-resistant Staphylococcus aureus (MRSA) had cefoxitin resistance, and among these MRSA isolates, 3 were resistant to vancomycin with the MIC technique. The antibiotic imipenem was the most effective against both Gram-positive and Gram-negative bacteria, and gentamicin was effective against Gram-negative bacteria.CONCLUSIONS: The present study confirmed the high prevalence of multidrug-resistant pathogens in diabetic foot ulcers. It is necessary to evaluate the different microorganisms infecting the wound and to know the antibiotic susceptibility patterns of the isolates from the infected wound. This knowledge is crucial for planning treatment with the appropriate antibiotics, reducing resistance patterns, and minimizing healthcare costs.

Novas bio-estratégias para a destoxificação de ocratoxina A utilizando bactérias do ácido láctico

A ocorrência de bolores micotoxigénicos pertencentes aos géneros Aspergillus, Penicillium e Fusarium em alimentos para consumo Humano e animal, tem um impacto importante sobre a saúde pública e constitui também um importante problema económico. Isto é devido à síntese por este tipo de fungos filamentosos de metabolitos altamente tóxicos conhecidos como micotoxinas. A maioria das micotoxinas são substâncias cancerígenas, mutagénicas, neurotóxicas e imunossupressoras, sendo a ocratoxina A (OTA) uma das mais importantes. A OTA é uma micotoxina, tóxica para os animais e Humanos principalmente devido às suas propriedades nefrotóxicas. Alguns grupos de bactérias gram positivas nomeadamente as bactérias do ácido láctico (BAL) são capazes de controlar o crescimento de fungos, melhorando e aumentando a vida útil de muitos produtos fermentados e, assim, reduzir os riscos para a saúde provocados pela exposição às micotoxinas. Algumas BAL são, também, capazes de destoxificar certas micotoxinas. Em trabalhos anteriores do nosso grupo foi observada a biodegradação da OTA por estirpes de Pediococcus parvulus isoladas de vinhos do Douro. Assim, com este trabalho, pretendeu-se compreender com maior detalhe o processo de biodegradação da OTA pelas referidas estirpes e identificar quais as enzimas que estão associadas à sua biodegradação. Para atingir este objetivo utilizaram-se algumas ferramentas ioinformáticas (BLAST, CLUSTALX2, CLC Sequence Viewer 7, Finch TV), desenharam-se primers específicos e realizaram-se PCR específicos para os genes envolvidos. Através da utilização de ferramentas de bioinformática, foi possível identificar várias proteínas que pertencem à família das carboxipeptidases e que podem eventualmente participar no processo da degradação da OTA, tais como D-Ala-D-Ala carboxipeptidase serínica e carboxipeptidase membranar. Estas BAL podem desempenhar um papel importante na destoxificação da OTA, sendo as carboxipeptidases uma das enzimas envolvidas na sua biodegradação.

A stable phage lysin (Cpl-1) dimer with increased antipneumococcal activity and decreased plasma clearance.

Bacteriophages (phages) produce endolysins (lysins) as part of their lytic cycle in order to degrade the peptidoglycan layer of the infected bacteria for subsequent release of phage progeny. Because these enzymes maintain their lytic and lethal activity against Gram-positive bacteria when added extrinsically to the cells, they have been actively exploited as novel anti-infectives, sometimes termed enzybiotics. As with other relatively small peptides, one issue in their clinical development is their rapid inactivation through proteolytic degradation, immunological blockage and renal clearance. The antipneumococcal lysin Cpl-1 was shown to escape both proteolysis and immunological blockage. However, its short plasma half-life (20.5 min in mice) may represent a shortcoming for clinical usefulness. Here we report the construction of a Cpl-1 dimer with a view to increasing both the antipneumococcal specific activity and plasma half-life of Cpl-1. Dimerisation was achieved by introducing specific cysteine residues at the C-terminal end of the enzyme, thus favouring disulphide bonding. Compared with the native monomer, the constructed dimer demonstrated a two-fold increase in specific antipneumococcal activity and a ca. ten-fold decrease in plasma clearance. As several lysins are suspected to dimerise on contact with their cell wall substrate to be fully active, stable pre-dimerised enzymes may represent a more efficient alternative to the native monomer.