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PubMed2002

[Lymphocyte apoptosis associated to infections].

During infections, the presence of lymphocyte apoptosis both in peripheral blood and in lymphatic organs has been described. This kind of programmed cell death can be either induced by host control mechanisms aimed at eliminating infected lymphocytes and/or retaining immune system homeostasis, or by the pathogen in order to complete its life cycle, spreading the infection and/or suppressing the immune response. Thus, apoptosis has advantages and disadvantages for the host depending on the pathogen life cycle and/or the specificity of the lymphocyte population affected. Identification of the mechanisms involved in autoreactive or pathogen-specific lymphocyte apoptosis could lead to strategies designed to interfere immunologically or pharmacologically in favor of the host.

Apoptosis↗
PubMed2004

16S rRNA gene based analysis of Enterobacter sakazakii strains from different sources and development of a PCR assay for identification.

BACKGROUND: E. sakazakii is considered to be an opportunistic pathogen, implicated in food borne diseases causing meningitis or enteritis especially in neonates and infants. Cultural standard identification procedures for E. sakazakii include the observation of yellow pigmentation of colonies and a positive glucosidase activity. Up to now, only one PCR system based on a single available 16S rRNA gene sequence has been published for E. sakazakii identification. However, in our hands a preliminary evaluation of this system to a number of target and non-target strains showed significant specificity problems of this system. In this study full-length 16S rRNA genes of thirteen E. sakazakii strains from food, environment and human origin as well as the type strain ATCC 51329 were sequenced. Based on this sequence data a new specific PCR system for E. sakazakii was developed and evaluated. RESULTS: By phylogenetic analysis of the new full-length 16S rRNA gene sequence data obtained we could show the presence of a second phylogenetic distinct lineage within the E. sakazakii species. The newly developed 16S rRNA gene targeting PCR system allows identification of E. sakazakii strains from both lineages. The assay's ability to correctly identify different E. sakazakii isolates as well as to differentiate E. sakazakii from other closely related Enterobacteriaceae species and other microorganisms was shown on 75 target and non-target strains. CONCLUSION: By this study we are presenting a specific and reliable PCR identification system, which is able to correctly identify E. sakazakii isolates from both phylogenetic distinct lines within the E. sakazakii species. The impact of this second newly described phylogenetic line within the E. sakazakii species in view of clinical and food safety aspects need further investigation.

Bacterial Typing Techniques↗
PubMed2006

PCR amplification of species specific sequences of 16S rDNA and 16S-23S rDNA intergenic spacer region for identification of Streptococcus phocae.

Streptococcus phocae, a bacterial pathogen of seals, could reliably be identified by PCR amplification using oligonucleotide primers designed according to species specific segments of the previously sequenced 16S rRNA gene and the 16S-23S rDNA intergenic spacer region of this species. The PCR mediated assay allowed an identification of S. phocae isolated from harbor and gray seals and from Atlantic salmons. No cross-reaction could be observed with 13 different other streptococcal species and subspecies and with Lactococcus garvieae strains investigated for control purposes.

Animals↗
PubMed2004

Comparative proteome analysis of cellular proteins extracted from highly virulent Francisella tularensis ssp. tularensis and less virulent F. tularensis ssp. holarctica and F. tularensis ssp. mediaasiatica.

Francisella tularensis is the causative agent of the zoonotic disease tularemia. Four subspecies of this pathogen, namely ssp. tularensis, mediaasiatica, holarctica, and novicida are spread throughout the northern hemisphere. Although there are marked variations in their virulence to mammals, the subspecies are difficult to identify as they are closely genetically related. We carried out the comparative proteome analysis of cellular extracts from isolates representing the highly virulent subspecies tularensis, and the less virulent subspecies mediaasiatica and holarctica in order to identify new diagnostic markers and putative factors of virulence. We identified 27 protein spots that were either specifically present or at significantly higher abundance in ssp. tularensis strains, 22 proteins in ssp. mediaasiatica strains, and 26 proteins in ssp. holarctica strains. Subspecies tularensis-specific proteins might represent putative virulence factors. Of 27 identified tularensis-specific spots 17 represented charge and mass variants of proteins occurring in other subspecies, 7 spots were found to be present at higher abundance, and 3 spots were specifically present in tularensis strains. Amongst them, PilP protein, as a component necessary for the biogenesis of the type IV pilus, virulence and adhesion factor for many human pathogen, was identified. Furthermore, the identification of additional 27 proteins common for ssp. tularensis and mediaasiatica, and 19 proteins shared by ssp. mediaasiatica and holarctica documented apparent closer genetic similarity between ssp. tularensis and mediaasiatica.

Bacterial Proteins↗
PubMed2005

Proteomics-based consensus prediction of protein retention in a bacterial membrane.

The availability of complete bacterial genome sequences allows proteome-wide predictions of exported proteins that are potentially retained in the cytoplasmic membranes of the corresponding organisms. In practice, however, major problems are encountered with the computer-assisted distinction between (Sec-type) signal peptides that direct exported proteins into the growth medium and lipoprotein signal peptides or amino-terminal membrane anchors that cause protein retention in the membrane. In the present studies, which were aimed at improving methods to predict protein retention in the bacterial cytoplasmic membrane, we have compared sets of membrane-attached and extracellular proteins of Bacillus subtilis that were recently identified through proteomics approaches. The results showed that three classes of membrane-attached proteins can be distinguished. Two classes include 43 lipoproteins and 48 proteins with an amino-terminal transmembrane segment, respectively. Remarkably, a third class includes 31 proteins that remain membrane-retained despite the presence of typical Sec-type signal peptides with consensus signal peptidase recognition sites. This unprecedented finding indicates that unknown mechanisms are involved in membrane retention of this class of proteins. A further novelty is a consensus sequence indicative for release of certain lipoproteins from the membrane by proteolytic shaving. Finally, using non-overlapping sets of secreted and membrane-retained proteins, the accuracy of different signal peptide prediction algorithms was assessed. Accuracy for the prediction of protein retention in the membrane was increased to 82% using a majority-vote approach. Our findings provide important leads for future identification of surface proteins from pathogenic bacteria, which are attractive candidate infection markers and potential targets for drugs or vaccines.

Amino Acid Sequence↗
PubMed2002

Identification and characterization of hydrophobic Escherichia coli virulence proteins by liquid chromatography-electrospray ionization mass spectrometry.

Virulence of enterotoxicogenic Escherichia coli is mediated by rodlike, rigid, highly hydrophobic proteins designated fimbriae or colonization factors (CFs). More than 20 different colonization factors have been described so far using predominantly immunological and genetic methods. To characterize these hydrophobic proteins by liquid chromatography-mass spectrometry (LC-MS), different methodologies were explored. A novel LC-MS method was developed using hexafluoroisopropanol to maintain the hydrophobic proteins in solution. In addition, these proteins were digested with cyanogen bromide and peptide mapping by LC-MS was established. This technique was particularly useful in identification of closely related CFs. Both LC-MS and peptide mapping methodologies were found to be useful in characterizing highly hydrophobic CFs of E. coli. To search for molecular weights of mature proteins in the National Center for Biotechnology Information (NCBI) database, a new feature was developed and its applicability tested. The identification of a class of pathogenic virulence proteins, either intact or digested, is possible with molecular weight database searching.

Bacterial Proteins↗
PubMed1991

Newly documented antimicrobial activity of quinolones.

The improved antimicrobial activity of newer fluoroquinolones and novel applications recently found for the drugs already marketed are reviewed. Several new compounds are more active against gram-positive bacteria than the presently marketed fluoroquinolones. WIN 57273, the most potent compound in vitro on a weight basis, is 16 to 128 times more active than ciprofloxacin against various staphylococci, streptococci, Enterococcus spp., Corynebacterium spp., Listeria monocytogenes and Bacillus spp. BMY 40062, PD 117558, PD 127391, sparfloxacin, temafloxacin and tosufloxacin also show enhanced in vitro efficacy against these species. These drugs also possess increased activity against various anaerobes, notably Clostridium perfringens, Clostridium difficile and the Bacteroides fragilis group. Mycobacterium tuberculosis, rapidly growing mycobacteria other than Mycobacterium chelonae, and Mycobacterium leprae are often susceptible to quinolones displaying bactericidal activity which is potentially useful for curing difficult-to-treat mycobacteriosis. In addition, a number of new products, notably those containing a cyclopropyl group, are more active than reference fluoroquinolones against Mycobacterium leprae. Sparfloxacin, BMY 40062 and WIN 57273 compare favorably with older fluoroquinolones in the killing of intracellular Legionella spp., and several of the newer compounds have greater antichlamydial potency. Improved antibacterial activity has also been found against Mycoplasma hominis, Ureaplasma urealyticum, Acinetobacter spp. and Pseudomonas maltophilia. By contrast, the newer quinolones have similar or less activity against Pseudomonas aeruginosa and Enterobacteriaceae. Recently, pefloxacin, ofloxacin and ciprofloxacin were found to be active against protozoa, including Plasmodium spp., Trypanosoma cruzi and Leishmania donovani, but not against Toxoplasma gondii. In the near future, more specific research testing unusual pathogens may lead to the identification of quinolones with more selective activity.

4-Quinolones↗
PubMedDate not supplied

Diagnosis of tuberculosis and other diseases caused by mycobacteria.

The adequate diagnosis and treatment of tuberculosis depends on many events, including rapid pathogen detection, patient isolation, species identification, and drug susceptibility testing. Well trained staff, using state-of-the-art technology, are necessary in the mycobacteriology laboratory to produce timely results that are necessary for the patients' care and public health measures. Mycobacteriology laboratories still play a pivotal role in the control of tuberculosis, which is especially true in view of the spread of multidrug-resistant tuberculosis. One way to optimize diagnostic efforts in spite of limited financial resources might be to sort and allocate specimens according to a system of priorities, e.g., diagnostic versus follow-up specimens. A "fast track" program for tuberculosis testing, which should be established as part of a dynamic diagnostic network, should focus on the highly infectious patient population. Collaboration between clinicians and mycobacteriologists remains the basis of dynamic diagnostic teamwork. Immediate screening of smears for acid-fast-bacilli in patients suspected of tuberculosis, followed by immediate processing of smear-positive specimens using modern mycobacteriological technology, should be given high priority. Diagnosis of disease due to nontuberculous mycobacteria can be difficult. Nontuberculous mycobacteria are commonly found in nature, and assessment as to whether a nontuberculous mycobacterium isolate is clinically significant can be a difficult task.

Humans↗
PubMed2002

clap1, a gene encoding a copper-transporting ATPase involved in the process of infection by the phytopathogenic fungus Colletotrichum lindemuthianum.

A screen for insertional mutants of Colletrichum lindemuthianum, the causative agent of common bean anthracnose, led to the identification of a non-pathogenic, lightly colored transformant. This mutant is unable to induce disease symptoms on intact or wounded primary leaves of seedlings and plantlets of Phaseolus vulgaris. In vitro, it exhibits normal vegetative growth, sporulation and conidial germination, but the cultures remain beige instead of becoming black. Microscopic examination revealed that this mutant forms fewer appressoria than the wild-type strain, and these are misshapen and poorly melanized. Molecular analyses indicated that the mutagenic plasmid had targeted clap1, a gene encoding a putative copper-transporting ATPase sharing 35% identity with the human Menkes and Wilson proteins and the product of the CCC2 gene of Saccharomyces cerevisiae. Complementation of the non-pathogenic beige mutant with a wild-type allele of clap1 restored both pathogenicity and pigmentation. Conversely, replacement of the wild-type allele with a disrupted clap1 gene gave rise to non-pathogenic beige transformants. Compared with the wild-type strain, extracts from clap1 mutants were found to have very low levels of phenol oxidase activity. These observations suggest that the clap1 gene product may be involved in the pathogenicity of C. lindemuthianum strains because of its role in delivering copper to secreted cuproenzymes, such as the phenol oxidases that mediate the polymerization of 1,8-dihydroxynaphthalene to melanin.

Adenosine Triphosphatases↗
PubMed1990

Detection of DNA from infectious laryngotracheitis virus by colourimetric analyses of polymerase chain reactions.

A combination of the polymerase chain reaction and a novel ELISA-type DNA colourimetric assay (developed from studies with a retrovirus from man) was used in a preliminary study to detect DNA from avian infectious laryngotracheitis virus. The method is sensitive, specific and easy to perform. Since it can be readily adapted for the detection of DNA from other sources it could be useful for the identification of a variety of pathogens from other species of veterinary importance.

Animals↗
PubMed1995

Genomic fingerprinting Acinetobacter baumannii: amplification of multiple inter-repetitive extragenic palindromic sequences.

Acinetobacter species are important nosocomial pathogens. A rapid and sensitive identification system, capable of providing strain identity at the genetic level, is required to identify outbreak strains and facilitate the early implementation of infection control procedures. Repetitive extragenic palindromic (REP) elements, have been identified in numerous bacteria and these genomic sequences provide useful targets for DNA amplification. A method for amplifying inter-REP DNA sequences, REP-multiple arbitrary amplicon profiling (REP-MAAP), is described and applied to 29 Acinetobacter baumannii from clinical samples. Amplified polymorphic DNA patterns were demonstrated for all isolates and those displaying identical REP-MAAP patterns were considered identical at the genetic level. In the spring of 1993, 10 intensive care unit patients had endotracheal colonization with A. baumannii (five with REP-MAAP I and five with REP-MAAP II patterns). These findings suggested nosocomial transmission of organisms which was terminated by standard infection control measures. No further A. baumannii were detected until the winter of 1993 when isolates of different REP-MAAP groups emerged, suggesting that factors other than nosocomial transmission were implicated.

Acinetobacter↗
PubMed1994

Polymerase chain reaction and its applications: special emphasis on its role in embryo sexing.

The polymerase chain reaction (PCR) has developed into one of the most promising methods for in vitro enzymatic amplification of DNA and has found widespread application in DNA cloning, sequencing and mutagenesis related studies. This innovative technique can selectively amplify a single target DNA molecule a billion-fold in a span of a few hours. Amplification of specific DNA sequences by PCR is useful in identification of sex, novel genes, pathogens and diseases. PCR has facilitated the establishment of evolutionary relationships among species and in revealing structural intricacies of single cells. In this article we review some of the major advances and applications of PCR, especially, its role in embryo sexing.

Journal Article↗
PubMed2026

Multimodal computational framework resolves B cell maturation in autoimmunity and ageing.

Identification of the origin of pathogenic immune cells is crucial for therapeutic interventions and diagnosis but pseudotime methods struggle to trace immune cells accurately. Current trajectory inference methods for B cell development and response in health and disease either ignore or underutilize antigen receptor sequence information, limiting their ability to resolve developmental pathways, particularly for pathogenic populations. Widely used methods such as Monocle 3 reconstruct developmental paths from transcriptomic similarity alone, discarding the features from immune receptors. Dandelion has combined the immune receptor features with transcriptomics but it struggles to simulate the trajectory path of B cells. Here we present ClonoTrace, a computational framework that integrates BCR sequence features with transcriptomic trajectory inference through gated fusion of multimodal embeddings. In fetal B cell development and germinal centre development, ClonoTrace demonstrates closer concordance with the canonical reference ordering than Monocle 3 and Dandelion. Applied to systemic lupus erythematosus, ClonoTrace indicates a memory B cell extrafollicular maturation route alongside the naïve B cell route, accompanied by induction of ZEB2 with a concomitant decline of BACH2 along the trajectory, as a candidate alternative route to pathogenic double negative 2 B cells (DN2) in systemic lupus erythematosus (SLE) patients. In healthy ageing, ClonoTrace resolved three candidate age-related B cell maturation routes, from naïve, IgM+ memory and switched-memory B cells, each passing through a DN2-associated transcriptional state that is ordered before age-associated B cells along the inferred trajectory. ClonoTrace's fate probability algorithm indicated that IgM+ memory B cell to ABC transition as the leading candidate age-associated transition, which may be distinct from SLE DN2 maturation. ClonoTrace provides a generalizable framework for receptor-informed trajectory inference, describing candidate developmental routes of pathogenic B cell populations in autoimmunity and ageing.

Humans↗
PubMed2006

Functional and structural characterization of Spl proteases from Staphylococcus aureus.

Staphylococcus aureus is the major cause of nosocomial infections world-wide, with increasing prevalence of community-acquired diseases. The recent dramatic increase in multi-antibiotic resistance, including resistance to the last-resort drug, vancomycin, together with the lack of an effective vaccine highlight the need for better understanding of S.aureus pathogenicity. Comparative analysis of available bacterial genomes allows for the identification of previously uncharacterized S.aureus genes with potential roles in pathogenicity. A good example is a cluster of six serine protease-like (spl) genes encompassed in one operon, which encode for putative proteases with similarity to staphylococcal glutamylendopeptidase (V8 protease). Here, we describe an efficient expression system for the production of recombinant SplB and SplC proteases in Escherichia coli, together with structural and functional characterization of the purified enzymes. A unique mechanism of cytoplasm protection against activity of misdirected SplB was uncovered. Apparently, the co-translated signal peptide maintains protease latency until it is cleaved by the signal peptidase during protein secretion. Furthermore, the crystal structure of the SplC protease revealed a fold resembling that of the V8 protease and epidermolytic toxins. Arrangement of the active site cleft and substrate-binding pocket of SplC explains the mechanism of enzyme latency and suggests that some Spl proteases possess restricted substrate specificity similar to that of the V8 protease and epidermolytic toxins.

Amino Acid Sequence↗
PubMed2005

Antimicrobial activity of the bufadienolides marinobufagin and telocinobufagin isolated as major components from skin secretion of the toad Bufo rubescens.

The increase in the emergence of antibiotic-resistant microorganisms and difficult to treat infections caused by these pathogens stimulate research aiming the identification of novel antimicrobials. Skin secretion of amphibian contains a large number of biologically active compounds, including compounds that performance defense mechanisms against microorganisms. In the present work, two antimicrobial bufadienolides, telocinobufagin (402.1609 Da) and marinobufagin (400.1515 Da), were isolated from skin secretions of the Brazilian toad Bufo rubescens. The specimens were collected in Brasilia (Distrito Federal, Brazil), the skin secretions extracted by electric stimulation, and submitted to purification by RP-HPLC. The molecular structure and mass determination were done by (1)H and (13)C NMR and mass spectrometry data, respectively. The antimicrobial activity was performed by liquid growth inhibition against Staphylococcus aureus and Escherichia coli. The minimum inhibitory concentrations of telocinobufagin and marinobufagin were, respectively, 64.0 and 16.0 microg/mL for E. coli and both 128 microg/mL for S. aureus. Besides the antimicrobial activity both bufadienolides promoted an increase of the contraction force in isolated frog ventricle strips.

Amphibian Venoms↗
PubMed2000

Detection and characterisation of parasites causing emerging zoonoses.

Understanding the epidemiology of zoonotic parasitic infections is dependent upon the availability of accurate and sensitive diagnostic techniques. The development of molecular diagnostic methods, particularly those utilising PCR for the detection of zoonoses will contribute greatly to the identification and control of these pathogens, by increasing the speed of diagnosis, specificity and sensitivity, reproducibility and ease of interpretation. Molecular characterisation studies allow us to distinguish between closely related infectious agents and to document the patterns of transmission of 'strains' and species within populations. This will allow precise determinations to be made about the aetiological agent, its characteristics and the source of infection. This review focuses on recent detection and characterisation techniques for both emerging and re-emerging parasite zoonoses.

Animals↗
PubMed1992

Community-acquired pneumonia.

The aetiology of community-acquired pneumonia is reviewed, and the identification of the most likely pathogens, based on clinical presentation, is discussed. By far the major pathogen in community-acquired pneumonia is Streptococcus pneumoniae; the relative frequency of other pathogens, and particularly the atypical pneumonias caused by Mycoplasma and Legionella spp., will depend on local epidemiological factors. The diagnostic tests to confirm diagnosis and subsequent treatment of these infections are reviewed.

Anti-Bacterial Agents↗