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PubMed1994

A simple procedure to differentiate ailA and ailNA gene variants among human pathogenic Yersinia enterocolitica strains.

The identification and differentiation of the two variants of the ail gene, ailA from the more virulent American serotypes (08, 013a, 13b, 018, 020, 021) and ailNA from the less virulent non-American serotypes (03, 04, 05, 06, 09, 027 and 07, 8) was studied in a panel of 32 Yersinia enterocolitica human pathogenic isolates. A 444 bp fragment corresponding to the ail gene was amplified using a PCR procedure in all tested strains. Subsequent digestion of the PCR product by Rsal and by HaeIII endonucleases, provide electrophoretic patterns that clearly discriminate ailA and ailNA variants. This non-radioactive and reliable procedure allows large clinical and epidemiological studies, and could be proposed to survey the spread of virulent clones.

Base Sequence↗
PubMed1998

Phenotypic, genotypic and pathogenic variation among streptomycetes implicated in common scab disease.

This study examines the diversity of streptomycetes causing potato scab using phenotypic identification, 16S rRNA sequence, and pathogenicity measurements. Pathogenicity did not correlate well with taxonomic identification, nor with phylogenetic position. This result suggests that neither phenotypic nor 16S characteristics are diagnostic for scab-causing strains. Additionally, phenotypic characterizations were not fully consistent with phylogenetic relationships. This incongruency highlights the difficulties of taxonomy within the streptomycetes and affirms the need for better integration of genetic and phenotypic characters in the systematics of this group.

DNA, Bacterial↗
PubMed1991

[Effects of antibiotics and anti-bacterial components of preparations for local treatment of suppurative wounds on pathogens of odonto- genic infections].

The results of identification and sensitivity assay of 156 strains of pathogens causing odontogenic infections are presented. In the sensitivity assay antibacterial drugs were used. 42.3 percent of the strains were obligate anaerobes belonging to Bacteroides, Fusobacterium, Peptococcus, Peptostreptococcus, Veillonella and Actinomyces. Significant differences in the microbial sensitivity to the drugs used for general and local therapy were detected. There was observed high sensitivity of the obligate anaerobes to gramicidin (0.02 micrograms/ml), nitazol (10 micrograms/ml), levomycetin and tetracycline (60 micrograms/ml). Antiseptics such as dioxidine and chlorhexidine used locally showed satisfactory results. The above mentioned drugs and especially levomycetin were also rather active against facultative organisms in associations of pathogens causing odontogenic infections: Bacillus coagulans, B. licheniformis, Pseudomonas sp., Acinetobacter calcoaceticus, Staphylococcus sp. and Streptococcus sp.

Anti-Bacterial Agents↗
PubMed2005

[Antigenome technology: rapid and comprehensive identification and validation of antigens as putative components in vaccines against bacterial pathogens].

The resolution of complete genome sequences of many pathogens has dramatically expanded our perspectives in developing novel vaccines by increasing both the speed and scale of target identification. Genomics-based technologies with their significant advantages can be applied to most pathogens and have exploited genome sequence information in alliance with adjunct technologies, including bioinformatics, expression analyses (microarrays, in vivo expression technologies), random mutagenesis or protein/peptide based selection methods (proteomics and immunoselection using expression libraries). A novel approach that combines the advantages of full genome coverage and serological antigen identification has been developed in the laboratory of the authors for the identification of protein antigens from bacterial pathogens. This technology has been applied to several bacterial pathogens and, as a result, defined the most relevant antigenic proteins that are targeted by the human immune system, including their antibody binding sites (epitopes). This catalogue of antigens is termed the antigenome and the novel technology offers an integrated approach for antigen validation in order to select the most promising candidates for the development of subunit vaccines against the targeted bacterial diseases. Using the antigenome technology, novel protective antigens had been discovered from several important human pathogens.

Antigens, Bacterial↗
PubMed2002

PCR-based assays for the fish pathogen Aeromonas salmonicida. I. Evaluation of three PCR primer sets for detection and identification.

In an effort to develop a rapid diagnostic test for the fish pathogen Aeromonas salmonicida, the performance of 2 polymerase chain reaction (PCR) primer sets (AP and PAAS) targeting the fish pathogen A. salmonicida and 1 PCR primer set (MIY) targeting A. salmonicida subsp. salmonicida were evaluated. Initially, the PCR assays were used to screen purified DNA extracted from 308 A. salmonicida isolates. The AP and PAAS PCR tests were demonstrated to be 100% specific for the species A. salmonicida and did not cross-react with any of the non-target organisms (bacterial species other than A. salmonicida) used in this study. The combined sensitivity of the AP and PAAS tests was 99.4% and offered the best coverage in terms of identifying the target organism. The MIY PCR appeared to be 100% sensitive and specific for A. salmonicida subsp. salmonicida. Studies with tissues, spiked with known quantities of bacteria, were conducted to determine the lower detection limit of the PCR tests, and then the ability of these PCR tests to detect A. salmonicida in experimentally infected salmonids was assessed.

Aeromonas↗
PubMed2003

Avian reservoirs and zoonotic potential of the emerging human pathogen Helicobacter canadensis.

A polyphasic identification approach was used to investigate the taxonomic position of Campylobacter-like isolates recovered from barnacle geese (Branta leucopsis) and Canada geese (Branta candensis). Seven strains were selected from a collection of 21 isolates and analyzed by extensive phenotypic testing; four strains were characterized by 16S rRNA gene sequence analysis. The results clearly identified the bird isolates as Helicobacter canadensis, recently described as an emerging human pathogen. This is the first report of an animal reservoir for this organism and of its presence in Europe and confirms the zoonotic potential of H. canadensis.

Animals↗
PubMed1988

Occurrence and clinical importance of the pathogenic serogroup O: 5, 27 of Yersinia enterocolitica in the Federal Republic of Germany and methods for its serological and bacteriological identification.

Despite its world-wide distribution, the pathogenic serogroup O: 5, 27 of Yersinia enterocolitica (Y. e.) has scarcely been considered in the Federal Republic of Germany. In the 1980 to 1986 period, a total of 74 strains from patients was identified at the National Reference Centre for Salmonella, Hamburg. The clinical manifestations of the cases was not different from those associated with Y. e. O: 3 and O: 9 infections. Six out of 8 patients from whom serum samples were available, developed moderate agglutination titres (1:80-1:1280). Only 8 of the 74 strains were negative in in vitro virulence tests. A list of biochemical tests is given which permit a differentiation of the pathogenic serogroup O: 5, 27 from the apathogenic Y. e. O: 5 which is widely distributed in the environment. Serological studies revealed different H antigens in both serogroups. The O antigen factor 27, and a subfactor O: 5(2) which is typical of Y. e. serogroup O: 5, are further differential markers; the production of both factor sera is described. Besides serogroups O: 3 and O: 9, Y. e. O: 5, 27 must be considered as an aetiological agent of human yersiniosis in the Federal Republic of Germany.

Adolescent↗
PubMed1997

The nephritogenic immune response.

Recent insights into the etiopathogenesis of nephritogenic immune responses are derived primarily from experimental models of systemic and organ-specific autoimmunity. Genetic analyses and immune-related gene ablation studies indicate that multiple independent mechanisms determine disease susceptibility. However, full characterization of proximal immunologic events in many diseases awaits identification of the renal antigens recognized by nephritogenic lymphocytes. Advances in characterization of effector mechanisms include epitope mapping of several putative pathogenic glomerular antigens and identification of novel pathways of immune-mediated tissue injury, including those involved in glomerular-tubulointerstitial communication and tubulointerstitial fibrosis. Finally, successful interruption of signal transduction pathways and transforming growth factor-beta 1 blockade by gene therapy suggest novel approaches to therapeutic intervention in immunologic renal injury.

Animals↗
PubMed2000

[Rapid and reliable detection of multiresistent Staphylococcus aureus (MRSA) by multiplex PCR].

BACKGROUND AND OBJECTIVE: Staphylococci are widespread pathogens and are frequently associated with nosocomial infections. Many hospitals struggle with increasing amounts of methicillin-resistant Staphylococcus aureus (MRSA) which are "multiresistant" against all betalactam antibiotics. Often, applicable antibiotics for treatment are only glycopeptides like vancomycin and teicoplanin. In addition, MRSA infected patients require expensive intensive isolation measures and strict hygiene. To efficiently prevent dissemination of these pathogens rapid and reliable identification and a close collaboration between clinicians and microbiologists are required. The purpose of our study was to set up a rapid and reliable identification procedure for MRSA by the amplification of specific gene determinants by PCR in order to to efficiently support therapy and eradication of the pathogen. METHODS: 153 strains of staphylococci isolated from in-patients of the hospital of the Justus-Liebig University of Giessen were examined. The femB gene was used to differentiate between Staphylococcus aureus (S. aureus) and coagulase-negative staphylococci (CNS), a gene which allows the species-specific identification of methicillin-resistant (MRSA) and -susceptible S. aureus (MSSA). Additionally, MRSA harbor the mecA gene encoding methicillin-resistance, which is absent in MSSA strains. RESULTS: Using a multiplex PCR with femB and mecA gene-specific oligonucleotides MRSA strains were unequivocally detected within 3 hours. The femB gene was detected in all 102 strains of S. aureus but in none of the 51 CNS. The mecA determinant was detected in 12 S. aureus. Among these, 11 strains were phenotypically methicillin-resistant and one strain was susceptible. The methicillin-resistance of this particular mecA-positive/methicillin-susceptible strain (cryptic MRSA) was inducible by cultivation on agar plates supplemented with flucloxacillin. CONCLUSIONS: The described method specifically detects S. aureus and identifies phenotypical and cryptic MRSA. These cryptic MRSA are of particular relevance since they are undetectable using common phenotypically based detection methods. It is conceivable that the methicillin resistance of these strains is induced under antibiotic therapy with flucloxacillin and that the mec-encoded feature of methicillin-resistance can be transferred to previously methicillin-susceptible strains. Using the reliable detection of these strains by PCR, failure of flucloxacillin therapy is avoidable.

Bacterial Proteins↗
PubMed2000

Avian infectious bronchitis virus.

Infectious bronchitis virus (IBV) is prevalent in all countries with an intensive poultry industry, with the incidence of infection approaching 100% in most locations. Vaccination is only partially successful due to the continual emergence of antigenic variants. At many sites, multiple antigenic types are simultaneously present, requiring the application of multiple vaccines. Although many countries share some common antigenic types, IBV strains within a geographic region are unique and distinct, examples are Europe, the United States of America and Australia. Measures to restrict the introduction of exotic IBV strains should therefore be considered. Infectious bronchitis has a significant economic impact; in broilers, production losses are due to poor weight gains, condemnation at processing and mortality, whilst in laying birds, losses are due to suboptimal egg production and downgrading of eggs. Chickens and commercially reared pheasants are the only natural hosts for IBV. Other species are not considered as reservoirs of IBV. The majority of IBV strains cause tracheal lesions and respiratory disease with low mortality due to secondary bacterial infections, primarily in broilers. Nephropathogenic strains, in addition to tracheal lesions, also induce prominent kidney lesions with mortality of up to 25% in broilers. Strains of both pathotypes infect adult birds and affect egg production and egg quality to a variable degree. Infected chicks are the major source of virus in the environment. Contaminated equipment and material are a potential source for indirect transmission over large distances. Virus is present in considerable titres in tracheal mucus and in faeces in the acute and recovery phases of disease, respectively. Virus spreads horizontally by aerosol (inhalation) or ingestion of faeces or contaminated feed or water. The virus is highly infectious. Clinical signs will develop in contact chicks within 36 h and in nearby sheds within one to two days. Infection is resolved within fourteen days with a rise in antibody titres. In a small number of chicks, latent infection is established with subsequent erratic shedding of virus for a prolonged period of time via both faeces and aerosol. Movement of live birds should be considered as a potential source for the introduction of IBV. Isolation and identification of IBV is needed for positive diagnosis. The preferred method of isolation is to passage a sample in embryonating specified-pathogen-free chicken eggs. Identification is either by monoclonal antibody based enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction. Virus neutralisation test in tracheal organ culture is the best method for antigenic typing. Continual use of live vaccines complicates diagnosis since no simple diagnostic tool can differentiate a field from a vaccine strain. Nucleotide sequencing of the S1 glycoprotein is the only method to discriminate between all IBV strains. Serology is also complicated by continual use of live vaccines. For surveillance purposes, ELISA is the method of choice, regardless of the antigenic type of IBV involved. The assay is used to monitor the response to vaccination, but field challenge can only be detected if flock antibody status is monitored continually. The antigenic type of a challenge strain involved cannot be ascertained by ELISA.

Animals↗
PubMed1975

Vaccination against bovine respiratory disease.

Vaccination is but one element in a control programme for bovine respiratory disease. Its laboratory study can be divorced from the others but its field application cannot. The problems associated with the development of effective vaccines fall into two broad groups: multiplicity and ubiquity of pathogens and secondly the identification of the crucial elements in an immune response. Agricultural systems which experience annual outbreaks of respiratory disease attributable to the same pathogen in cattle of specific age have the choice of using passive or active immunity of minimal valency. In the majority of systems the cause and timing of an outbreak cannot be predicted and therefore multivalent vaccines are required. Both inactivated and modified live products are available for use against the well-known pathogens. Their relative advantages hinge on the significance attributed to the ability to stimulate the production of particular immunoglobulins at specific body sites and the persistence of the responses. The widely held view that success requires the stimulation of secretory antibodies by intranasal administration of living vaccines is not universally accepted. An assessment of their protective value is not easily made because of the difficulty of reproducing an adequate field challenge in the laboratory. The measurement of serological responses and virus shedding times following challenge are of limited value as alternatives.

Animals↗
PubMed1976

Entomopathogens: ecological manipulation of natural associations.

The control of insect pests with entomopathogens is unique, in that naturally occurring host-pathogen relations are manipulated to the benefit of man: protecting agricultural crops and forests or controlling insect vectors of disease. The isolation and identification of a virulent pathogen is the initial step in the development of a potential control agent. Production of the pathogen in adequate quantities must be possible either in vivo (insects) or in vitro (artificial medium). To insure usefulness, the pathogen must remain viable in the formulated form and after application in the field. Since inactivation rather than persistence is a problem, the pathogens must be formulated, protected, and applied to insure satisfactory pest control action. Studying the natural host--pathogen interactions will be necessary in order to manipulate the pathogen effectively, by introducing it at the most opportune time in the life cycle of the target pest. Generally, insect pathogens are more selective than conventional pesticides; this will limit their use and industrial development. Development, at least in part, by the public sector may be necessary and desirable. The most promising areas for the use of pathogens are in integrated pest management and in situations where pests have developed resistance to chemical control.

Animals↗
PubMed2002

Vaccines of the future: from rational design to clinical development. 17-19 October 2001, Paris, France.

The Institut Pasteur (France) sponsors and organises a series of Euroconferences on important topics in biology, medicine and environmental sciences which facilitate a large exchange of ideas between basic and applied scientists from the Institut Pasteur, other academic institutions, and pharmaceutical companies. This Euroconference focused on the most recent advances currently affecting the way vaccine development is approached. The identification of new target antigens for vaccination purposes is presently facilitated by the availability of genome sequences from a growing number of important pathogens and tumours. The identification of T-cell epitopes and the subsequent optimisation of these target sequences allows the design of vaccines which are significantly improved with respect to their capacity to stimulate cellular immunity. In parallel, various antigen formulations, adjuvants and delivery systems are being developed and tested with the aim of eliciting broad immune responses in humans. These new orientations capitalise on recent major advances in the understanding of the use of therapeutic vaccines and expands on their use from being solely profilactic. Specific issues related to the development of therapeutic vaccines directed against diseases such as cancer or chronic infectious disease were presented.

Antigens, Neoplasm↗
PubMed2025

Rapid and accurate sepsis diagnostics via a novel probe-based multiplex real-time PCR system.

Sepsis is a critical clinical emergency that requires prompt diagnosis and intervention. Its prevalence has increased due to the aging population and increased antibiotic resistance. Early identification and the use of innovative technologies are crucial for improving patient outcomes. Modern methodologies are needed to minimize the turnaround time for diagnosis and improve outcomes. Rapid diagnostic tests and multiplex PCR are effective but have limitations in identifying a range of pathogens and target genes. Our study evaluated two novel probe-based multiplex real-time PCR systems: the SEPSI ID and SEPSI DR panels. These systems can quickly identify bacterial and fungal pathogens, alongside antibiotic resistance genes. The assays cover 29 microorganisms (gram-negative bacteria, gram-positive bacteria, yeast, and mold species), alongside 23 resistance genes and four virulence factors. A streamlined workflow uses 2 µL of broth from positive blood cultures (BCs) without nucleic acid extraction and provides results in approximately 1 h. We present the results from an evaluation of 228 BCs and 22 isolates previously characterized by whole-genome sequencing. In comparison to the reference methods, the SEPSI ID panel demonstrated a sensitivity of 96.88%, a specificity of 100%, and a PPV of 100%, whereas the SEPSI DR panel showed a sensitivity of 97.8%, a PPV of 89.7%, and a specificity of 96.7%. Both panels also identified additional pathogens and resistance-related targets not detected by conventional methods. This assay shows promise for rapidly and accurately diagnosing sepsis. Future studies should validate its performance in various clinical settings to enhance sepsis management and improve patient outcomes.IMPORTANCEWe present a new diagnostic method that enables the quick and precise identification of pathogens and resistance genes from positive blood cultures, eliminating the need for nucleic acid extraction. This technique can also be used on fresh pathogen cultures. It has the potential to greatly improve treatment protocols, leading to better patient outcomes, more responsible antibiotic use, and more efficient management of healthcare resources.

Humans↗
PubMed2005

Genomic and bioinformatics analysis of HAdV-7, a human adenovirus of species B1 that causes acute respiratory disease: implications for vector development in human gene therapy.

Human adenovirus serotype 7 (HAdV-7) is a reemerging pathogen identified in acute respiratory disease (ARD), particularly in epidemics affecting basic military trainee populations of otherwise healthy young adults. The genome has been sequenced and annotated (GenBank accession no. ). Comparative genomics and bioinformatics analyses of the HAdV-7 genome sequence provide insight into its natural history and phylogenetic relationships. A putative origin of HAdV-7 from a chimpanzee host is observed. This has implications within the current biotechnological interest of using chimpanzee adenoviruses as vectors for human gene therapy and DNA vaccine delivery. Rapid genome sequencing and analyses of this species B1 member provide an example of exploiting accurate low-pass DNA sequencing technology in pathogen characterization and epidemic outbreak surveillance through the identification, validation, and application of unique pathogen genome signatures.

Adenovirus Infections, Human↗
PubMed1993

Mapping quantitative trait loci (QTLs) for resistance to Gibberella zeae infection in maize.

The basic prerequisite for an efficient breeding program to improve levels of resistance to pathogens in plants is the identification of genes controlling the resistance character. If the response to pathogens is under the control of a multilocus system, the utilization of molecular markers becomes essential. Stalk and ear rot caused by Gibberella zeae is a widespread disease of corn: resistance to G. zeae is quantitatively inherited. Our experimental approach to understanding the genetic basis of resistance to Gibberella is to estimate the genetic linkage between available molecular markers and the character, measured as the amount of diseased tissue 40 days after inoculation of a suspension of Fusarium graminearum, the conidial form of G. zeae, into the first stalk internode. Sensitive and resistant parental inbreds were crossed to obtain F1 and F2 populations: the analysis of the segregation of 95 RFLP (restriction fragment length polymorphism) clones and 10 RAPD (random amplified polymorphic DNA) markers was performed on a population of 150 F2 individuals. Analysis of resistance was performed on the F3 families obtained by selfing the F2 plants. Quantitative trait loci (QTL) detection was based either on analysis of regression coefficients between family mean value and allele values in the F2 population, or by means of interval mapping, using MAPMAKER-QTL. A linkage map of maize was obtained, in which four to five genomic regions are shown to carry factors involved in the resistance to G. zeae.

Chromosome Mapping↗
PubMed1998

Isolation of an amoeba naturally harboring a distinctive Legionella species.

There are numerous in vitro studies documenting the multiplication of Legionella species in free-living amoebae and other protozoa. It is believed that protozoa serve as host cells for the intracellular replication of certain Legionella species in a variety of environmental settings. This study describes the isolation and characterization of a bacterium initially observed within an amoeba taken from a soil sample. In the laboratory, the bacterium multiplied within and was highly pathogenic for Acanthamoeba polyphaga. Extracellular multiplication was observed on buffered charcoal yeast extract agar but not on a variety of conventional laboratory media. A 16S rRNA gene analysis placed the bacterium within the genus Legionella. Serological studies indicate that it is distinct from previously described species of the genus. This report also describes methods that should prove useful for the isolation and characterization of additional Legionella-like bacteria from free-living amoebae. In addition, the characterization of bacterial pathogens of amoebae has significant implications for understanding the ecology and identification of other unrecognized bacterial pathogens.

Amoeba↗
PubMed2005

Genomics of clostridial pathogens: implication of extrachromosomal elements in pathogenicity.

The recently decoded genomes of the major clostridial toxin-producing pathogens Clostridium perfringens, Clostridium tetani, Clostridium botulinum and Clostridium difficile have provided a huge amount of new sequence data. Recent studies have focused on the identification and investigation of pathogenic determinants and the regulatory events governing their expression. The sequence data revealed also the genomic background of virulence genes, as well as the contribution of extrachromosomal elements to a pathogenic phenotype. This has generated new insights in clostridial pathogenesis - and will continue to do so in the future - and has deepened our understanding of the anaerobic lifestyle of clostridial species.

Clostridioides difficile↗