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PubMed2000

Phenotypic and genotypic comparisons of 23 strains from the Bacillus cereus complex for a selection of known and putative B. thuringiensis virulence factors.

Sixteen Bacillus thuringiensis, four Bacillus cereus and three Bacillus anthracis isolates were screened for a selection of known and putative B. thuringiensis virulence factors. PCR primers were designed to detect genes for phosphatidylcholine specific phospholipase C, phosphatidylinositol specific phospholipase C, immune inhibitor A, vegetative insecticidal protein 3A, a protein proposed to be involved in capsule synthesis, a newly identified Ser/Thr kinase homologue and enterotoxin entS. Motility, the presence of flagella, haemolysis, chitinase and lecithinase production were also evaluated. The widely varying profiles of the 23 strains from the complex provide a pool of different genotypes that can help to identify factors involved in pathogenicity.

Bacillus anthracis↗
PubMed2005

Study on morphology, pathogenicity, and genetic variability of Beauveria bassiana isolates obtained from Boophilus microplus tick.

Fifty isolates of Beauveria bassiana (Balsamo) Vuillemin, 1912 (Ascomycota: Clavicipitaceae) were analyzed by morphology, for their pathogenic potential to Boophilus microplus (Canestrini, 1887) (Acari: Ixodidae) larvae, and by Random Amplified Polymorphic DNA-Polymerase Chain Reaction technique. Morphological analysis demonstrated that isolates present characteristics compatible to those described for B. bassiana in the literature. Virulence test demonstrated that all isolates present lethal effect on larvae and that the lethal concentration varies among isolates. The most virulent isolate was the only one obtained from human infection, which was also the only isolate presenting synnemata. The study on genetic variability among the isolates allowed the identification of 23 electrophoretic profiles. The established groupings suggest that most of the isolates obtained from B. microplus of the same locality present low genetic variation. In this way, the data in the present study will contribute to a meticulous characterization of these B. bassiana isolates.

Animals↗
PubMed2005

Phase and size variable surface-exposed proteins in equine genital mycoplasmas.

Mycoplasma equigenitalium and Mycoplasma subdolum have been associated with infertility, endometritis, vulvitis and abortions in mares, and with reduced fertility and balanoposthitis in stallions. Despite their role in equine genital disorder, determinants of virulence and pathogenesis as well as factors provoking specific host immune responses have not been identified, so far. To establish the major immunogenic components of Mycoplasma (M.) equigenitalium and M. subdolum, antigen profiles of their type strains as well as 30 clinical isolates were compared by SDS-PAGE and immunoblot analysis using hyperimmune rabbit sera and equine sera from clinical cases. To define the major protein antigens of both mycoplasma species, detergent-phase fractionation with Triton X-114 was performed. Western blot analysis of 30 clinical isolates revealed a high similarity of their overall antigen profile with only slight differences. In contrast, monospecific polyclonal antibodies raised against detergent-phase proteins of the two mycoplasma species identified three prominent proteins (pST17, pST42, and pET45) undergoing variation in expression and size among clinical and clonal isolates.

Animals↗
PubMed1986

Extraintestinal Campylobacter jejuni and Campylobacter coli infections: host factors and strain characteristics.

To determine whether extraintestinal isolates of Campylobacter jejuni and Campylobacter coli are the consequence of unusual host or bacterial characteristics, we studied clinical and bacteriologic features of 24 extraintestinal infections. Common serotypes and auxotypes were present among the extraintestinal isolates. Gastrointestinal isolates were more susceptible to normal human serum than were the systemic isolates; however, the ranges overlapped considerably. Predispositions to systemic spread were present in 52% of patients with extraintestinal infections; isolates from these patients were more often (73%) serum sensitive than were isolates from patients without predispositions (9%; P = .002). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, no specific protein band was associated with serum resistance, and all isolates of C. jejuni and C. coli had rough-type lipopolysaccharide profiles. Serum susceptibility was inversely correlated with carbohydrate or ketodeoxyoctonate (KDO) fraction of cell weight and directly correlated with KDO:carbohydrate ratio. Our results suggest that either host defects or specific bacterial virulence characteristics, such as serum resistance, possibly related to length of lipopolysaccharide side chain, may be responsible for extraintestinal infections due to C. jejuni and C. coli.

Adolescent↗
PubMed2003

EST mining and functional expression assays identify extracellular effector proteins from the plant pathogen Phytophthora.

Plant pathogenic microbes have the remarkable ability to manipulate biochemical, physiological, and morphological processes in their host plants. These manipulations are achieved through a diverse array of effector molecules that can either promote infection or trigger defense responses. We describe a general functional genomics approach aimed at identifying extracellular effector proteins from plant pathogenic microorganisms by combining data mining of expressed sequence tags (ESTs) with virus-based high-throughput functional expression assays in plants. PexFinder, an algorithm for automated identification of extracellular proteins from EST data sets, was developed and applied to 2147 ESTs from the oomycete plant pathogen Phytophthora infestans. The program identified 261 ESTs (12.2%) corresponding to a set of 142 nonredundant Pex (Phytophthora extracellular protein) cDNAs. Of these, 78 (55%) Pex cDNAs were novel with no significant matches in public databases. Validation of PexFinder was performed using proteomic analysis of secreted protein of P. infestans. To identify which of the Pex cDNAs encode effector proteins that manipulate plant processes, high-throughput functional expression assays in plants were performed on 63 of the identified cDNAs using an Agrobacterium tumefaciens binary vector carrying the potato virus X (PVX) genome. This led to the discovery of two novel necrosis-inducing cDNAs, crn1 and crn2, encoding extracellular proteins that belong to a large and complex protein family in Phytophthora. Further characterization of the crn genes indicated that they are both expressed in P. infestans during colonization of the host plant tomato and that crn2 induced defense-response genes in tomato. Our results indicate that combining data mining using PexFinder with PVX-based functional assays can facilitate the discovery of novel pathogen effector proteins. In principle, this strategy can be applied to a variety of eukaryotic plant pathogens, including oomycetes, fungi, and nematodes.

Algal Proteins↗
PubMed2003

Identification of peptides that mimic the pertussis toxin binding site on bovine fetuin.

The introduction of acellular pertussis vaccines has greatly enhanced the safety profile of vaccines to prevent whooping cough. Pertussis toxin (Ptx) is one component produced by Bordetella pertussis that is contained in all of these vaccines, either in combination with other known pertussis virulence factors or as the sole pertussis component, combined with tetanus and diphtheria toxoids. A hydrogen peroxide toxoid of Ptx has been shown to be efficacious in preventing pertussis infections in a mass vaccination trial and is presently licensed in the United States and Europe (B. Trollfors, J. Taranger, T. Lagergard, L. Lind, V. Sundh, G. Zackrisson, C. U. Lowe, W. Blackwelder, and J. B. Robbins, N. Engl. J. Med. 333:1045-1050, 1995). The industrial production of Ptx can be performed through the cultivation of B. pertussis in well-defined growth media, in which the components can be well characterized and their origins can be documented. Once the bacteria are removed from the culture, Ptx can be isolated from the supernatant and purified by using the technique described by Sekura et al. (R. D. Sekura, F. Fish, C. R. Manclark, B. Meade, and Y. L. Zhang, J. Biol. Chem. 258:14647-14651, 1983). The only drawback of this procedure, which combines two affinity chromatography steps, one with Blue Sepharose and a second with matrix-bound bovine fetuin (BF), is the source and purity of the BF. Concern about vaccine preparations that may possibly risk contamination by material associated with bovine spongioform encephalopathy has continued to increase. We thus sought a replacement for the BF affinity chromatography and, more specifically, for the glycosidic moiety on BF. We describe here the identification of a seven-amino-acid peptide that mimics the glycosidic moiety on BF to which Ptx binds. Furthermore, we have constructed an affinity column containing this peptide that can be used to replace BF in Ptx purification. Finally, we used the X-ray crystallographic structure of Ptx bound to the oligosaccharide moiety of BF as a scaffold and replaced the oligosaccharide with the peptide.

Amino Acid Sequence↗
PubMed1990

Electrophoretic analysis of the surface components of autoagglutinating surface array protein-positive and surface array protein-negative Aeromonas hydrophila and Aeromonas sobria.

The protein and lipopolysaccharide (LPS) compositions of 10 autoagglutinating Aeromonas hydrophila and Aeromonas sobria strains were studied; one group consisted of five serogroup O:11 strains that contained an S layer, while a second group was composed of diverse serogroups that were S layer negative by transmission electron microscopy. All serogroup O:11 strains were found to contain a predominant 52,000- to 54,000-molecular-weight protein that was present on both whole-cell and outer membrane protein profiles; this protein was found to be glycine extractable under low-pH (pH 4) conditions and was identified as the surface array protein. LPS analysis revealed that all O:11 strains exhibited homogeneous-length O-polysaccharide side chains characterized primarily by two or three major bands. In contrast, S-layer-negative autoagglutinating strains of other serogroups lacked this predominant surface array protein, and silver stain analysis of LPS indicated that such profiles mainly consisted of core antigens and were deficient in or devoid of O-polysaccharide side chains. These collective results offer potential explanations for observed differences between these two groups in virulence, disease spectrum, and pathogenic properties.

Aeromonas↗
PubMed1970

Lack of nucleotide sequence relationship among the temperate bacteriophage SPO2, Bacillus subtilis, and virulent bacteriophages beta 3 and beta 22.

Radiolabeled phage SPO2 fragments were tested for the abiity to form interspecies deoxyribonucleic acid (DNA) duplexes with DNA from Bacillus subtilis and from phages beta3 and beta22. No reassociation, above control values, occurred between the DNA of phage SPO2 and that of its host or either of the virulent B. subtilis phages. It appears that extensive nucleotide sequence similarities between portions of host and phage DNA species are not necessary for the formation of the lysogenic state. The thermal elution profile of reassociated SPO2 DNA exhibited a normal distribution, indicating no heterogeneity in base composition. This differs from temperate enterophages whose DNA molecules contain regions of differing base composition.

Bacillus subtilis↗
PubMed2006

Microarray analysis of human epithelial cell responses to bacterial interaction.

Host-pathogen interactions are inherently complex and dynamic. The recent use of human microarrays has been invaluable to monitor the effects of various bacterial and viral pathogens upon host cell gene expression programs. This methodology has allowed the host response transcriptome of several cell lines to be studied on a global scale. To this point, the great majority of reports have focused on the response of immune cells, including macrophages and dendritic cells. These studies revealed that the immune response to microbial pathogens is tailored to different microbial challenges. Conversely, the paradigm for epithelial cells has--until recently--held that the epithelium mostly served as a relatively passive physical barrier to infection. It is now generally accepted that the epithelial barrier contributes more actively to signaling events in the immune response. In light of this shift, this review will compare transcriptional profiling data from studies that involved host-pathogen interactions occurring with epithelial cells. Experiments that defined both a common core response, as well as pathogen-specific host responses will be discussed. This review will also summarize the contributions that transcriptional profiling analysis has made to our understanding of bacterial physio-pathogensis of infection. This will include a discussion of how host transcriptional responses can be used to infer the function of virulence determinants from bacterial pathogens interacting with epithelial mucosa. In particular, we will expand upon the lessons that have been learned from gastro-intestinal and oral pathogens, as well as from members of the commensal flora.

Animals↗
PubMed2006

Methicillin-resistant Staphylococcus aureus in children, Taiwan.

Highly virulent community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) with Panton-Valentine leukocidin (PVL) is common worldwide. Using antimicrobial drug susceptibility testing, staphylococcal cassette chromosome mec typing, exotoxin profiling, and pulsed-field gel electrophoresis typing, we provide evidence that supports the relationship between nasal strains of PVL-positive MRSA and community-acquired disease.

Adolescent↗
PubMed2000

Acquisition of microbiological data to enhance food safety.

The routine acquisition and archiving of microbiological data is undertaken for two reasons. The first is the development of historical microbiological profiles of foods, ingredients, or processes in order to determine or verify that microorganisms of concern are being controlled to the level desired. The second reason is data concerning the pathogenicity or virulence of foodborne pathogens and their behavior in foods in order to develop strategies and criteria for assuring microbiological safety. Both types of microbiological data are essential to effective food safety programs. A firm understanding of the uses and limitations of both is essential to correct acquisition, interpretation, and use of such data.

Databases, Factual↗
PubMed2001

Correlation between Congo red binding and contact haemolysin production in Shigella species.

Haemolytic strains of Shigella dysenteriae type 1, Shigella flexneri, Shigella boydii and Shigella sonnei cultured on Congo red agar produced pigmented colonies (Pcr+) whereas nonhaemolytic strains produced white colonies and did not bind Congo red (Pcr-). S. flexneri-1 haemolysin negative mutant (lacking plasmid) of haemolysin positive prototroph also did not bind Congo red and produced nonpigmented colonies. Among the twelve strains of Shigella included in this study, the characteristics of Congo red binding, plasmid profile and haemolytic activity appeared to be correlated. Congo red binding occurred comparatively more by haemolysin-producing strains. Congo red binding can be used as a quick and reliable method for virulence traits of pathogens, including haemolysin activity.

Bacterial Adhesion↗
PubMed1999

[Epidemiological characteristics and virulence factor of verotoxin-producing Eschericia coli O121:H19 isolated in Akita Prefecture in July 1997].

Epidemiological characteristics and virulence factors of VTEC O121:H19 strains isolated in July 1997 from a 15 year old female and a 20 year old male patient suffering from bloody diarrhea and severe abdominal pain were examined. The 2 VTEC O121:H19 isolates showed identical antibiotic susceptibility patterns, biochemical characteristics and plasmid profile while slight differences were observed in their Xba I and Not I PFGE patterns, suggesting that closely related 2 VTEC O121:H19 strains evoked the sporadic infectious cases in July 1997. The 2 VTEC O121:H19 isolates, as well as VTEC O157:H7, possessed eaeA gene and a ca. 60 MDa plasmid which hybridised with CVD 419 probe and produced enterohemolysin. In addition, the VTEC O121:H19 isolates produced almost the same amount of VT-2 in vitro as VTEC O157:H7 did. These results suggested that VTEC O121:H19 possesed the virulence factor comparable to that of VTEC O157:H7. Incidence, molecular epidemiology and infectious source of VTEC O121:H19 in this country have not been sufficiently understood. Antiserum for E. coli serogroup O121 should be manufactured to clarify the epidemiology of the highly virulent VTEC strain.

Adolescent↗
PubMed2005

Distribution of antimicrobial resistance and virulence-related genes among Brazilian group B streptococci recovered from bovine and human sources.

In the present report we describe the characteristics of 189 antimicrobial-resistant Streptococcus agalactiae isolates from bovine (38 isolates) and human (151 isolates) sources. All the strains were resistant to tetracycline (TET), and 16 (8.5%) were also resistant to erythromycin, corresponding to 23.7% of the TET-resistant bovine isolates and 4.6% of the TET-resistant human isolates. The tet(O), erm(B), and mreA resistance-related genes, as well as the bca and scpB virulence-related genes, were the most frequent among the bovine isolates, while the tet(M), erm(A), mreA, bca, lmb, and scpB genes were the most prevalent among the isolates from humans. Although a few major clusters were observed, pulsed-field gel electrophoresis results revealed a variety of profiles, reflecting the substantial genetic diversity among strains of this species isolated from either humans or bovines.

Animals↗
PubMed2005

Environmental and genetic regulation of the phosphorylcholine epitope of Haemophilus influenzae lipooligosaccharide.

In response to environmental signals in the host, bacterial pathogens express factors required during infection and repress those that interfere with specific stages of this process. Signalling pathways controlling virulence factors of the human respiratory pathogen, Haemophilus influenzae, are predominantly unknown. The lipooligosaccharide (LOS) outer core represents a prototypical virulence trait of H. influenzae that enhances virulence but also provides targets for innate and adaptive immunity. We report regulation of the display of the virulence-associated phosphorylcholine (PC) epitope on the LOS in response to environmental conditions. PC display is optimal under microaerobic conditions and markedly decreased under conditions of high culture aeration. Gene expression analysis using a DNA microarray was performed to begin to define the metabolic state of the cell under these conditions and to identify genes potentially involved in PC epitope modulation. Global gene expression profiling detected changes in redox responsive genes and in genes of carbohydrate metabolism. The effects on carbohydrate metabolism led us to examine the role of the putative H. influenzae homologue of csrA, a regulator of glycolysis and gluconeogenesis in Escherichia coli. A mutant containing an in-frame deletion of the H. influenzae csrA gene showed increased PC epitope levels under aerobic conditions. Furthermore, deletion of csrA elevated mRNA expression of galU, an essential virulence gene that is critical in generating sugar precursors needed for polysaccharide formation and LOS outer core synthesis. Growth conditions predicted to alter the redox state of the culture modulated the PC epitope and galU expression as well. The results are consistent with a multifactorial mechanism of control of LOS-PC epitope display involving csrA and environmental signals that coordinately regulate biosynthetic and metabolic genes controlling the LOS structure.

Aerobiosis↗
PubMed2000

[The niches and pathways of animal pathogens].

Infectious diseases are not a relict of the past but a topical phenomenon determined by complex evolution of the currently existing and constantly changing microbial agents and their hosts. With regard to abundance of species within the microbial kingdom and rate of its changes and development, it is difficult to predict the role of the microbial factor in mortality of humans and animals. The study and generalization of sequential similarities of microbial virulence factors after the completion of genome sequencing of principal pathogens can play a positive role in this direction. At present, molecular-genetic methods allow us to study the phylogenetic relationships of microbes and categorize them according to new criteria. The efficient control of diseases caused by microbes requires knowledge on their physiological and ecological niche from which they penetrate, in various ways, into the host organisms and, under suitable conditions, induce mass diseases. This process has several stages and, in the recent period, it is increasingly affected by human activities. The knowledge on all participants in this process, i.e. the microbe and its niche, factors of virulence and pathways of their dissemination, requires a scientifically based surveillance. Abundance and variability is characteristic for both microbial kingdom and microbial niche. Some identification of pathogenic properties of microorganisms and factors affecting their movement from their niche to the recipients results in activation of old classical diseases (e.g. plague, cholera, tuberculosis etc.) or emerging of new, so far unknown infections diseases ("emerging inf. disease"--EID), caused for example by lentiviruses, oncoviruses, filiviruses, bartonella, borrelia etc. This has provided the basis for establishment of new medical trends and approaches, such as "Emergency medicine" or "Travel medicine", expressing their purpose by their names. The control of existing or proposed infectious diseases in the 21st century (in which majority of factors such as urbanization, environmental factors, evolution of the microbial kingdom, will contribute to the persistence or "emergence" of new diseases) will be affected by the input of new knowledge in the field of molecular biology, such as introduction of biosensors, genetic tests, microchips, new generation of DNA vaccines, enteric vaccines and antibodies produced by transgenic animal bioreactors or plants, "customized" vaccines assessed for individual genetic profiles, etc. (Tab. 5, Ref. 21.)

Animals↗
PubMed1989

[Outer membranes of Francisella tularensis and their protein composition].

Four strains of the species Francisella tularensis were used in the present work: a live vaccine strain 15/10 and three virulent strains (503, Schu, 543) from three different subspecies. The bacterial membranes were prepared by the 0.5% N-laurylsarcosinate (Sarcosyl) treatment. These membranes were identified as the outer membranes by morphological, immunological and biochemical analyses. The outer membrane proteins contained up to 30-35 polypeptides with three dominant fractions having the 63, 48 and 41-43 kD molecular masses. Despite the significant similarity between the membranes protein profiles there were some quantitative and qualitative differences between the three variants of Francisella tularensis in polypeptides compositions and patterns.

Bacterial Outer Membrane Proteins↗
PubMed2025

Ergosterol-depleted clinical isolates of Nakaseomyces glabratus can develop multi-drug resistance without apparent fitness and virulence defects.

OBJECTIVES: Nakaseomyces glabratus (formerly Candida glabrata) is a leading cause of invasive candidiasis and rapidly develops antifungal drug resistance during treatment. An increasing number of clinical isolates shows reduced susceptibility to echinocandins and azoles, leaving amphotericin B (AMB) as a last therapeutic option. Resistance of N. glabratus to this drug is rare and its underlying mechanisms are still not fully understood. Here, we describe two independent multidrug resistant (MDR) bloodstream isolates displaying resistance to AMB and anidulafungin (ANF) as well as a reduced susceptibility to azoles. METHODS: Whole-genome sequencing and sterol profiling were performed on nine clinical N. glabratus isolates which were resistant to ANF and displayed resistance or low susceptibility to fluconazole (FLU) and AMB. The transcriptional response of reference strain CBS138 and an AMBR+ANFR isolate was analyzed by RNA-seq. Furthermore, PDR1 was deleted in the latter isolate to examine its influence on efflux pump gene expression. Additionally, fitness and virulence of the AMBR+ANFR isolate were examined in growth assays and a Galleria mellonella infection model. RESULTS: Loss of function mutations in the genes ERG3 and ERG4 is linked to ergosterol depletion and AMB resistance. Ergosterol depletion also contributed to a Pdr1-mediated up-regulation of ERG and ABC transporter genes which was associated with low FLU susceptibility. The AMBR isolates displayed no fitness defects and one of them was fully virulent in a G. mellonella infection model. CONCLUSIONS: These findings demonstrate that ergosterol depletion in N. glabratus leads to AMB resistance without affecting fitness or virulence.

Journal Article↗