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PubMed2004

Pathogenicity islands in bacterial pathogenesis.

In this review, we focus on a group of mobile genetic elements designated pathogenicity islands (PAI). These elements play a pivotal role in the virulence of bacterial pathogens of humans and are also essential for virulence in pathogens of animals and plants. Characteristic molecular features of PAI of important human pathogens and their role in pathogenesis are described. The availability of a large number of genome sequences of pathogenic bacteria and their benign relatives currently offers a unique opportunity for the identification of novel pathogen-specific genomic islands. However, this knowledge has to be complemented by improved model systems for the analysis of virulence functions of bacterial pathogens. PAI apparently have been acquired during the speciation of pathogens from their nonpathogenic or environmental ancestors. The acquisition of PAI not only is an ancient evolutionary event that led to the appearance of bacterial pathogens on a timescale of millions of years but also may represent a mechanism that contributes to the appearance of new pathogens within a human life span. The acquisition of knowledge about PAI, their structure, their mobility, and the pathogenicity factors they encode not only is helpful in gaining a better understanding of bacterial evolution and interactions of pathogens with eukaryotic host cells but also may have important practical implications such as providing delivery systems for vaccination, tools for cell biology, and tools for the development of new strategies for therapy of bacterial infections.

Bacteria↗
PubMed2005

Detection of sexually transmitted pathogens in patients with hematospermia.

BACKGROUND: Although the current literature attributes most cases of hematospermia to an infectious agent, identification of the specific pathogens involved has been limited. OBJECTIVES: To determine the prevalence of different pathogens in patients presenting with hematospermia to our sexually transmitted disease clinic. METHODS: Between January 1999 and January 2000, 16 patients presented to our STD clinic with hematospermia after other noninfectious pathologies had been excluded by a referring physician. After obtaining informed consent, subjects completed a questionnaire addressing symptoms and sexual behavior. First-void urine samples, as well as genitourinary and serum specimens were tested for Chlamydia trachomatis, Ureaplasma urealyticum and herpes simplex virus. Standard bacterial cultures were also performed. RESULTS: Laboratory testing detected a pathogen in 12 of the 16 males presenting with hematospermia. The sexually transmitted pathogens detected were herpes simplex virus in 5 patients (42%), Chlamydia trachomatis in 4 (33%), Enterococcus fecalis in 2 (17%), and Ureaplasma urealyticum in 1 (8%). In all cases in which a pathogen was identified, the appropriate antimicrobial agent was administered. Symptoms resolved for each patient following antimicrobial therapy. During a 1 year follow-up, all 12 patients remained free of disease. CONCLUSIONS: Recent advances in microbiologic diagnostic techniques have facilitated the detection of pathogens in patients with hematospermia, thereby enhancing the efficacy of treatment.

Adolescent↗
PubMed2005

[Detection of sexually transmitted pathogens in patients with hematospermia].

BACKGROUND: Although the current literature attributes most cases of hematospermia to an infectious agent, identification of the specific pathogens involved has been limited. OBJECTIVE: To determine the prevalence of different pathogens, in patients presenting with hematospermia to our sexually transmitted disease clinic. PATIENTS AND METHODS: We retrospectively examined the laboratory results of 30 patients who presented to our clinic with hematospermia after other non-infectious pathologies had been excluded by a referring physician. Firstly, void urine samples as well as genitourinary and serum specimens were tested for Herpes simplex, Chlamydia trachomatis and Ureaplasma urealyticum. Standard bacterial cultures were also performed. RESULTS: In 12 of the 18 patients a possible pathogenic cause for the hematospermia was found. Evidence for Herpes simplex virus (Type 2) was found in 5 of the patients (16.6%), for Chlamydia trachomatis in 3 patients (10%) and for Ureaplasma urealyticum in 2 of the patients. Enterococcus fecalis was detected in 2 (6.6%) of the patients and in 1 patient Herpes simplex virus (Type 1) antigen and antibodies were detected. In all cases in which evidence of a pathogen was identified, the appropriate antimicrobial agent was administered, with recurrence in two patients only. CONCLUSIONS: Recent advances in microbiologic diagnostic techniques have facilitated the detection of pathogens in patients with hematospermia, thereby enhancing the efficacy of treatment.

Chlamydia trachomatis↗
PubMed1998

Rapid detection of Actinobacillus actinomycetemcomitans, Prevotella intermedia and Porphyromona gingivalis by multiplex PCR.

The identification of specific periodontal pathogens by conventional methods, mainly anaerobic cultivation, is difficult, time consuming and even sometimes unreliable. Therefore, a multiplex PCR method for simultaneous detection of Actinobacillus actinomycetemcomitans (A.a.), Porphyromona gingivalis (P.g.) and Prevotella intermedia (P.i.) was developed for rapid and easy identification of these specific bacterial pathogens in subgingival plaque samples. In this paper, there is a detailed description of the oligonucleotide primer selection, DNA extraction and PCR conditions and the sequencing of the amplified products. The locus chosen to be amplified is a highly variable region in the 16S ribosomal DNA. For the development of this technique ATCC cultures and pure cultures from subgingival plaque samples taken from periodontitis patients were used. As an internal positive control a recombinant plasmid was developed. This simple DNA extraction procedure and the DNA amplification and visualization of the amplified product permits the detection of the bacteria in a working day. Thus, this multiplex PCR method is a rapid and effective detection method for specific periodontal pathogens.

Aggregatibacter actinomycetemcomitans↗
PubMed1989

[Infectious complications in the early phase following kidney transplantation].

A retrospective analysis of 533 patients receiving kidney transplantation was performed to study the incidence of infection in the early postoperative period. Mostly localized in the lungs and renal system, bacterial complications arose in 133 patients. As compared with the unproblematic management of the urinary tract infections, 45 pulmonary infections were characterized by difficulties in diagnosis and treatment. Poor graft function was closely related to pulmonary infections: mean creatinine was 2.4 mg% (in patients without pneumonia - 1.5 mg%). Out of 45 patients with pneumonia, the graft failed in 16 patients. 6 patients died as a result of pneumonia. Rapid detection of the pathogenic organism is required, if necessary by invasive diagnosis. The administration of erythromycin before identification of the responsible pathogen may be indicated, in view of the fatal outcome in several patients subsequently diagnosed as having Legionella infection.

Adolescent↗
PubMed2000

rpoB gene analysis as a novel strategy for identification of spirochetes from the genera Borrelia, Treponema, and Leptospira.

Spirochetes are emerging pathogens for which culture and identification are partly unresolved. In fact, 16S rRNA-based sequencing is by far the most widely used PCR methodology that is able to detect such uncultivable pathogens. However, this assay actually has some limitations linked to potential problems of contamination, which hampers diagnosis. To circumvent this, we have devised a simple PCR strategy involving targeting of the gene encoding the RNA polymerase beta subunit (rpoB), a highly conserved enzyme. The complete sequence of the Leptospira biflexa (serovar patoc) rpoB gene was determined and compared with the published sequences for Borrelia burgdorferi and Treponema pallidum. From the resulting analysis, degenerate nucleotide primers were designed and tested for their ability to amplify a portion of the rpoB gene from various spirochetes. Using two different pairs of these primers, we succeeded in obtaining specific rpoB-amplified fragments for all members of the genera Leptospira, Treponema, and Borrelia tested and no other bacteria. Our findings may have significant implications for the development of a new tool for the identification of spirochetes, especially if clinical samples are contaminated or when the infecting strain is uncultivable.

Borrelia↗
PubMed1995

A specific oligonucleotide probe based on 5S rRNA sequences for identification of Vibrio anguillarum and Vibrio ordalii.

An oligonucleotide DNA probe based on 5S rRNA sequence data was constructed for the identification of the fish pathogens, Vibrio anguillarum and Vibrio ordalii. Specificity of the probe was tested in a colony blot hybridization assay. The respective probe was found to be specific for both V. anguillarum and V. ordalii. No cross hybridization was observed against other fish pathogens and the closely related Vibrionaceae genera. This specific probe may be useful for rapid identification of V. anguillarum and V. ordalii.

Base Sequence↗
PubMed1997

New directions in diagnostics.

BACKGROUND: Infectious diseases are still a significant clinical problem in children, and accurate identification of the causal pathogen plays an important role in clinical management. The availability of an etiologic diagnosis enables the clinician to make appropriate therapeutic decisions and to avoid the indiscriminate use of antibiotics. The availability of a microbiologic diagnosis and the susceptibility profile of the pathogen allows the prompt initiation of suitable antibiotic treatment. However, the usefulness of current culture and identification methods is limited by the time needed and by their sensitivity and specificity. Also some microorganisms are difficult or impossible to grow in the laboratory. OBJECTIVES: To review the newer and more rapid diagnostic techniques that are becoming available and consider their application in the diagnosis of specific infections. DISCUSSION: Immunoassays have many advantages and it is hoped that new optical immunoassays will overcome the problems of poor sensitivity. Nucleic acid amplification techniques have enormous potential in the diagnosis of infectious diseases because of their high specificity and sensitivity and the speed with which the results can be obtained. However, there are still a number of difficulties that must be overcome before these methods can be widely adopted for routine testing. These techniques may be particularly relevant for the rapid diagnosis of streptococcal pharyngitis, where throat culture is slow and beset by a number of factors which reduce its accuracy. Polymerase chain reaction methods have been developed for many respiratory pathogens, including Mycoplasma pneumoniae, Chlamydia pneumoniae and Mycobacterium tuberculosis, and are likely to play an increasingly important part in diagnosis. In bacterial meningitis culture is still the gold standard and molecular techniques have not yet been developed to the point where they can be used in routine diagnosis. Nucleic acid techniques are likely to be very valuable in the diagnosis of streptococcal pharyngitis and viral central nervous system infections in the near future.

Bacterial Infections↗
PubMed2024

Development of a clinical metagenomics workflow for the diagnosis of wound infections.

BACKGROUND: Wound infections are a common complication of injuries negatively impacting the patient's recovery, causing tissue damage, delaying wound healing, and possibly leading to the spread of the infection beyond the wound site. The current gold-standard diagnostic methods based on microbiological testing are not optimal for use in austere medical treatment facilities due to the need for large equipment and the turnaround time. Clinical metagenomics (CMg) has the potential to provide an alternative to current diagnostic tests enabling rapid, untargeted identification of the causative pathogen and the provision of additional clinically relevant information using equipment with a reduced logistical and operative burden. METHODS: This study presents the development and demonstration of a CMg workflow for wound swab samples. This workflow was applied to samples prospectively collected from patients with a suspected wound infection and the results were compared to routine microbiology and real-time quantitative polymerase chain reaction (qPCR). RESULTS: Wound swab samples were prepared for nanopore-based DNA sequencing in approximately 4 h and achieved sensitivity and specificity values of 83.82% and 66.64% respectively, when compared to routine microbiology testing and species-specific qPCR. CMg also enabled the provision of additional information including the identification of fungal species, anaerobic bacteria, antimicrobial resistance (AMR) genes and microbial species diversity. CONCLUSIONS: This study demonstrates that CMg has the potential to provide an alternative diagnostic method for wound infections suitable for use in austere medical treatment facilities. Future optimisation should focus on increased method automation and an improved understanding of the interpretation of CMg outputs, including robust reporting thresholds to confirm the presence of pathogen species and AMR gene identifications.

Humans↗
PubMed2006

Identification and functional expression of the pepper pathogen-induced gene, CAPIP2, involved in disease resistance and drought and salt stress tolerance.

A novel pathogen-induced gene, designated CAPIP2, was isolated from pepper leaves infected with Xanthomonas campestris pv. vesicatoria. CAPIP2:GFP fusion proteins were primarily localized in the cytoplasm. The CAPIP2 transcripts were constitutively expressed in the pepper leaves, flowers, and fruits, but were not detected in the stems and roots. CAPIP2 gene expression was induced strongly in the pepper leaves during pathogen infection, and also after exposure to abiotic elicitors and environmental stresses. Ectopic CAPIP2 expression in Arabidopsis was accompanied by the expression of Arabidopsis PR-1 and PDF1.2 genes. Overexpression of the CAPIP2 gene in Arabidopsis transgenic plants conferred enhanced resistance to Pseudomonas syringae pv. tomato DC3000. The CAPIP2 transgenic Arabidopsis also manifested increased tolerance to high salt, drought and oxidative stress during seed germination and seedling state. These results suggest that pepper CAPIP2 gene may function as a defense-related gene against both biotic and abiotic stresses.

Amino Acid Sequence↗
PubMed1998

Identification of ceramide-phosphorylethanolamine in oomycete plant pathogens: Pythium ultimum, Phytophthora infestans, and Phytophthora capsici.

Cellular lipids were extracted from three species of Oomycete plant pathogens (Pythium ultimum, Phytophthora infestans, and Ph. capsici) and analyzed via normal-phase high-performance liquid chromatography with flame-ionization detection. The most abundant polar lipids in each of the three species were the polar membrane lipids, phosphatidylethanolamine (PE), phosphatidylcholine, and a phosphosphingolipid that eluted soon after PE. Structural analysis via mass spectrometry and nuclear magnetic resonance spectrometry revealed that the phosphosphingolipid was ceramide phosphorylethanolamine (Cer-PE). The most abundant molecular species of Cer-PE in P. ultimum had a molecular weight of 670.5, contained an unusual 19-carbon branched triunsaturated sphingoid (C19-delta 4, 8, 10, 9-methyl long-chain base) and palmitic acid as the amide-linked fatty acid. The most abundant molecular species of Cer-PE in Ph. infestans had a molecular weight of 714.5, contained a common 16-carbon 1,3 di-OH sphingoid, and erucic (cis 13-docosenoic, C22-delta 13) acid as the amide-linked fatty acid. The Cer-PE in Ph. capsici comprised a mixture of each of the two molecular species found in P. ultimum and Ph. infestans.

Ceramides↗
PubMed2002

Identification of differentially expressed transcripts in the human pathogenic fungus Paracoccidioides brasiliensis by differential display.

Paracoccidioides brasiliensis is a dimorphic human pathogenic fungus that is the causal agent of paracoccidioidomycosis, a systemic disease that predominantly affects rural communities in South and Central America. Dimorphism is a common characteristic of systemic human pathogenic fungi. Here we describe the use of differential display (DD) to isolate and identify differentially expressed genes of P. brasiliensis, in the two cell types, yeast (Y) and mycelium (M), as well as at different time intervals during temperature-induced M to Y transition. Using two oligo-deoxythymidine-anchored primers combined with 10 arbitrary ones, we were able to detect the presence of at least 20 differentially transcribed cDNA fragments. Some of these fragments were further analysed by reverse-northern blot and northern blot in order to confirm their differential expression. The M32, M51 and M73 cDNA fragments were specific for the mycelial form of P. brasiliensis. Furthermore, we found two cDNA fragments (M-Y1 and M-Y2) that were upregulated during M-Y transition. This method was efficient and useful in the detection of differentially expressed genes in P. brasiliensis.

DNA, Complementary↗
PubMed2005

Blood-meal analysis for the identification of reservoir hosts of tick-borne pathogens in Ireland.

The results of analysis of blood-meal remnants in unfed nymphs, despite relatively low detection levels (49.4%, n = 322), support the conclusion from an earlier study that small rodents are relatively unimportant as reservoir hosts of B. burgdorferi s.l. in this particular area, and suggest that songbirds (Passeriformes) are the most significant hosts in this respect. Tick (Ixodes ricinus) abundance was greater in the present study, but the overall Borrelia burgdorferi s.l.-infection prevalence of nymphal ticks was the same (12.2%), and the relative proportions of the various Borrelia burgdorferi s.l. genospecies were similar. B. garinii and B. valaisiana were the most frequent, B. burgdorferi s.s the least frequent, and B. afzelii of intermediate frequency. An unusually high proportion of nymphs (39%) with multiple infections of different B. burgdorferi genospecies was detected, and Borrelia spp. related to relapsing-fever spirochetes were detected in Ireland for the first time. The results of the present study contribute to the validation of blood-meal analysis as a means of determining the host origin of certain pathogens in unfed questing ticks, and raise some questions concerning the extent of B. burgdorferi s.l. host specificity.

Animals↗
PubMed2006

Interpolated variable order motifs for identification of horizontally acquired DNA: revisiting the Salmonella pathogenicity islands.

MOTIVATION: There is a growing literature on the detection of Horizontal Gene Transfer (HGT) events by means of parametric, non-comparative methods. Such approaches rely only on sequence information and utilize different low and high order indices to capture compositional deviation from the genome backbone; the superiority of the latter over the former has been shown elsewhere. However even high order k-mers may be poor estimators of HGT, when insufficient information is available, e.g. in short sliding windows. Most of the current HGT prediction methods require pre-existing annotation, which may restrict their application on newly sequenced genomes. RESULTS: We introduce a novel computational method, Interpolated Variable Order Motifs (IVOMs), which exploits compositional biases using variable order motif distributions and captures more reliably the local composition of a sequence compared with fixed-order methods. For optimal localization of the boundaries of each predicted region, a second order, two-state hidden Markov model (HMM) is implemented in a change-point detection framework. We applied the IVOM approach to the genome of Salmonella enterica serovar Typhi CT18, a well-studied prokaryote in terms of HGT events, and we show that the IVOMs outperform state-of-the-art low and high order motif methods predicting not only the already characterized Salmonella Pathogenicity Islands (SPI-1 to SPI-10) but also three novel SPIs (SPI-15, SPI-16, SPI-17) and other HGT events. AVAILABILITY: The software is available under a GPL license as a standalone application at http://www.sanger.ac.uk/Software/analysis/alien_hunter CONTACT: gsv@sanger.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Algorithms↗
PubMed2005

Identification of a tomatinase in the tomato-pathogenic actinomycete Clavibacter michiganensis subsp. michiganensis NCPPB382.

The insertion site of a transposon mutant of Clavibacter michiganensis subsp. michiganensis NCPPB382 was cloned and found to be located in the gene tomA encoding a member of the glycosyl hydrolase family 10. The intact gene was obtained from a cosmid library of C. michiganensis subsp. michiganensis. The deduced protein TomA (543 amino acids, 58 kDa) contains a predicted signal peptide and two domains, the N-terminal catalytic domain and a C-terminal fibronectin III-like domain. The closest well-characterized relatives of TomA were tomatinases from fungi involved in the detoxification of the tomato saponin alpha-tomatine which acts as a growth inhibitor. Growth inhibition of C. michiganensis subsp. michiganensis by alpha-tomatine was stronger in the tomA mutants than in the wild type. Tomatinase activity assayed by deglycosylation of alpha-tomatine to tomatidine was demonstrated in concentrated culture supernatants of C. michiganensis subsp. michiganensis. No activity was found with the tomA mutants. However, neither the transposon mutant nor a second mutant constructed by gene disruption was affected in virulence on the tomato cv. Moneymaker.

Actinobacteria↗
PubMed1994

Identification of the gp67 gene of a baculovirus pathogenic to the spruce budworm, Choristoneura fumiferana multinucleocapsid nuclear polyhedrosis virus.

The baculovirus gp67 gene encodes a pH-dependent membrane fusion protein and has been identified in both Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) and Orygia pseudotsugata MNPV (OpMNPV). We have identified a homologous gene in the spruce budworm virus, Choristoneura fumiferana MNPV (CfMNPV). The CfMNPV gp67 gene is 79% identical to AcMNPV gp67 at the level of nucleotide sequence and 82% identical at the level of predicted amino acid sequence. As with OpMNPV and AcMNPV gp67, the CfMNPV gp67 protein is found exclusively in the budded virus phenotype of the baculovirus.

Amino Acid Sequence↗
PubMedDate not supplied

Identification of proteins in Encephalitozoon intestinalis, a microsporidian pathogen of immunocompromised humans: an immunoblotting and immunocytochemical study.

Microsporidia are unicellular and obligate intracellular spore-forming parasites. The spore inoculates the host cell with its non-motile infectious content, the sporoplasm, by way of the polar tube--the typical invasive apparatus of the microsporidian spore. Molecules involved in host cell invasion were investigated in Encephalitozoon intestinalis. Mouse polyclonal and monoclonal antibodies were raised against spore proteins and their reactivity was tested by Western-blotting and immunolocalization techniques, including electron and confocal microscopy. The antibodies thus generated could be divided into two major groups. One group reacted to the surface of the parasite at different developmental stages, mostly presporous stages and mature spores, whereas the other group recognized the polar tube. Of the antibodies reacting to the spore wall, one identified an exospore protein at 125 kDa while all others recognized a major doublet at 55-60 kDa, and minor proteins present at the surface of sporogonic stages and in the endospore. All antibodies recognizing spore wall proteins reacted also to the material forming septa in the parasitophorous vacuole. A major polar tube protein at 60 kDa was identified by another group of antibodies.

Animals↗
PubMed2006

Identification of genes from the obligate intracellular plant pathogen, Plasmodiophora brassicae.

Plasmodiophora brassicae is an intracellular pathogen that infects plants in the Brassicaceae family. Although an important pathogen group, information on the genomic makeup of the plasmodiophorids is almost completely lacking. We performed suppression subtractive hybridization (SSH) between RNA from P. brassicae-infected and uninfected Arabidopsis tissue, then screened 232 clones from the resulting SSH library. In addition, we used an oligo-capping procedure to screen 305 full-length cDNA clones from the infected tissue. A total of 76 new P. brassicae gene sequences were identified, the majority of which were extended to full length at the 5' end by the use of RACE amplification. Many of the unisequences were predicted to contain signal peptides for ER translocation. Although we located few sequences in total, these markedly increase available data from the plasmodiophorids, and provide new opportunities to examine plasmodiophorid biology. Our study also points towards the best methods for future plasmodiophorid gene discovery.

Brassica↗