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PubMed2006

Phylogenetic origins of African and Neotropical Beauveria bassiana s.l. pathogens of the coffee berry borer, Hypothenemus hampei.

A phylogenetic epidemiological study of Beauveria bassiana s.l. was conducted for African and Neotropical pathogens of the coffee berry borer (CBB), Hypothenemus hampei, based on inferences from two nuclear intergenic regions, EFutr and Bloc. CBB pathogens were distributed among four terminal clades, however, the majority of African and Neotropical isolates cluster in a well-supported monophyletic group, informally designated AFNEO_1. Although the relationship between African and Neotropical AFNEO_1 is unresolved, the majority of alleles detected were exclusive to either the African or the Neotropical populations. These fixed genetic differences suggest that their disjunction predates the world trade in coffee. Neotropical AFNEO_1 have a broad host range and CBB pathogens are intermixed phylogenetically with isolates from diverse indigenous insects. Several Neotropical AFNEO_1 isolates were isolated from coffee plants as epiphytes or endophytes, thus plants themselves may potentially serve as reservoirs of pathogens against their insect pests. Topological incongruence between the EFutr and Bloc phylogenies of Neotropical AFNEO_1 may signify that individuals within this population are recombining.

Africa↗
PubMed2006

Expression profiling of Botrytis cinerea genes identifies three patterns of up-regulation in planta and an FKBP12 protein affecting pathogenicity.

The ascomycete Botrytis cinerea is a broad-spectrum plant pathogen. Here, we describe the first macroarray transcriptomic study of the fungus in real-time infection conditions. Infection of Arabidopsis thaliana leaves by B.cinerea was monitored using macroarrays, containing 3032 genes. Variance analysis revealed that 7% of B.cinerea genes are differentially expressed during infection and allowed us to identify 27 genes significantly up-regulated in planta. Among them, two genes have already been associated with fungal pathogenicity, while eight genes have unidentified functions. The 27 genes were separated into three groups according to their expression profile. The first group showed maximal expression at the early stage following fungal penetration, the second one showed maximal expression at the outset of the colonization of plant leaves and the third group showed maximal expression when the colonization of plant leaves was completed. A gene of the last group (BcPIC5), which is homologous to FKBP12 proteins, was disrupted in order to determine its role in pathogenicity. At seven days post-inoculation, the lesions caused by the DeltaBcPIC5 mutant on bean leaves were reduced by 69% and did not further expand compared to the wild-type. These results confirm that transcriptomic analysis under infection conditions can be very valuable for the identification of fungal genes related to pathogenicity.

Arabidopsis↗
PubMed2005

Type III effector proteins from the plant pathogen Xanthomonas and their role in the interaction with the host plant.

Pathogenicity of Xanthomonas campestris pathovar (pv.) vesicatoria and most other Gram-negative bacterial plant pathogens largely depends on a type III secretion (TTS) system which is encoded by hypersensitive response and pathogenicity (hrp) genes. These genes are induced in the plant and are essential for the bacterium to be virulent in susceptible hosts and for the induction of the hypersensitive response (HR) in resistant host and non-host plants. The TTS machinery secretes proteins into the extracellular milieu and effector proteins into the plant cell cytosol. In the plant, the effectors presumably interfere with cellular processes to the benefit of the pathogen or have an avirulence activity that betrays the bacterium to the plant surveillance system. Type III effectors were identified by their avirulence activity, co-regulation with the TTS system and homology to known effectors. A number of effector proteins are members of families, e.g., the AvrBs3 family in Xanthomonas. AvrBs3 localizes to the nucleus of the plant cell where it modulates plant gene expression. Another family that is also present in Xanthomonas is the YopJ/AvrRxv family. The latter proteins appear to act as SUMO cysteine proteases in the host. Here, we will present an overview about the regulation of the TTS system and its substrates and discuss the function of the AvrRxv and AvrBs3 family members in more detail.

Amino Acid Sequence↗
PubMed2025

Rapid pan-microbial metagenomics for pathogen detection and personalised therapy in the intensive care unit: a single-centre prospective observational study.

BACKGROUND: Most clinical metagenomic studies do not provide rapid results, detect pathogens from all microbial kingdoms, or measure clinical impacts. We aimed to evaluate the feasibility, performance, and clinical impacts of a rapid pan-microbial respiratory metagenomic service for patients admitted to intensive care units (ICUs). METHODS: This was a single-centre observational study of a rapid metagenomics service that tests respiratory samples from ICU patients at Guy's and St Thomas' hospitals, London, UK, between Dec 5, 2023, and April 12, 2024. Testing used a previously published pan-microbial metagenomics workflow, which simultaneously detects bacteria, fungi, and DNA and RNA viruses; provides same-day preliminary results after 2 h; and provides final results after 24 h. Patients were included if they were aged 18 years or older, admitted to the ICU, had confirmed respiratory failure requiring supplemental oxygen or advanced airway support, and had at least one of the following: (1) clinical suspicion of lower respiratory tract infection based on clinical, biochemical, or radiological findings, (2) sepsis of unknown origin, and (3) concern from an intensive care physician regarding inflammatory pathology. Patients with a suspected or confirmed containment level three organism were excluded. The outcome was performance characteristics of the metagenomic test compared with routine diagnostic testing, detection of additional pathogens by metagenomics, change in antimicrobial prescribing within 24 h of testing, and initiation of immunomodulation. FINDINGS: We processed 114 samples (1-5 per day) from 74 patients (39 [53%] female and 35 [47%] male). 107 (94%) of 114 samples passed quality control, of which 101 (94%) provided same-day preliminary results. Bacteria were detected in 45 (43%) of 104 tested specimens, fungal organisms in 17 (16%) of 104 tested specimens, and viruses in 28 (34%) of 83 tested specimens. Sensitivity in lower respiratory tract samples after 24 h was 97% (95% CI 87-100) for bacteria, 89% (65-99) for fungi, and 89% (71-98) for viruses, with only one false positive for bacteria. Metagenomics identified 42 pathogens not detected by other tests in 32 (30%) of 107 samples. Antimicrobial therapy was changed after metagenomic results from 30 (28%) of 107 samples: 22 (21%) were de-escalated and eight (7%) were escalated. Metagenomics contributed to the initiation of immunomodulation in 15 (20%) of 74 patients for a range of inflammatory conditions. Pathogens with clinical significance to local infection control or national public health were found in ten (14%) of 74 patients, including three invasive Group A streptococci, two parvovirus B19, and one each of HIV-1, measles virus, Mycobacterium tuberculosis, Neisseria meningitidis, and Mycoplasma pneumoniae. INTERPRETATION: Respiratory metagenomics for ICU patients showed good performance and turnaround time, and diverse clinical and public health benefits. This ability to inform both personalised patient therapy and infectious disease surveillance needs evaluation in multicentre studies. FUNDING: None.

Humans↗
PubMed2004

Multiplex PCR allows simultaneous detection of pathogens in ships' ballast water.

There is enormous potential for global transfer of microorganisms, including pathogens, in ships' ballast water. We contend that a major advancement in the study of ballast-water microorganisms in particular, and of aquatic pathogens in general, will be expedited sample analysis, such as provided by the elegant technology of DNA microarrays. In order to use DNA microarrays, however, one must establish the appropriate conditions to bind target sequences in samples to multiple probes on the microarrays. We conducted proof-of-concept experiments to optimize simultaneous detection of multiple microorganisms using polymerase chain reaction (PCR) and Southern hybridization. We chose three target organisms, all potentially found in ballast water: a calicivirus, the bacterium Vibrio cholerae, and the photosynthetic protist Aureococcus anophagefferens. Here, we show simultaneous detection of multiple pathogens is possible, a result supporting the promising future use of microarrays for simultaneous detection of pathogens in ballast water.

Blotting, Southern↗
PubMed2004

Evaluation of lig-based conventional and real time PCR for the detection of pathogenic leptospires.

Leptospirosis is globally important infectious disease affecting almost all mammals. Pathogenic Leptospira encodes immunoglobulin-like protein (Lig) that is found to express only during infection. We report the development of conventional and real time PCR assays targeting lig genes of leptospires for the early diagnosis of leptospirosis. Sensitivity of the newly designed Lig1/Lig2 primers for conventional PCR was compared with previously published primers LP1/LP2 and G1/G2. G1/G2 primers amplified the target DNA from all the serovars including non-pathogenic Leptospira biflexa whereas LP1/LP2 and Lig1/Lig2 primers amplified only pathogenic leptospires. Diagnostic PCR assay was also developed for the detection of pathogenic Leptospira interrogans in urine samples. We obtained the highest sensitivity in PCR using our Lig1/Lig2 primers with a detection of 6 leptospires. A rapid and sensitive lig-based real time PCR assay was also developed with a detection range of 10-10(7) gene copies. To evaluate the early diagnosis for leptospirosis, we compared the culture with conventional and real time PCR for the detection of spirochetes in experimentally infected hamsters during a time-course study. Culture of infected hamster tissues detected the presence of leptospires from Day 2 of infection but not on the day of infection or Day 1, whereas conventional PCR and real time PCR detected the leptospires from the day of infection. Hence, conventional and real time PCR with lig primers would be a sensitive and rapid tool for early diagnosis of leptospirosis.

Animals↗
PubMed2004

Ice as a reservoir for pathogenic human viruses: specifically, caliciviruses, influenza viruses, and enteroviruses.

Hundreds of isolates of viable bacteria and fungi have been recovered from ancient ice and permafrost. Evidence supports the hypothesis that viral pathogens also are preserved in ice repositories, such as glaciers, ice sheets, and lake ice. Proof may depend upon narrowing the search by applying specific criteria, which would target candidate viruses. Such criteria include viral pathogens likely to occur in great abundance, likely to be readily transported into ice, and then participate in ongoing disease cycles suggestive of their having been deposited in and subsequently released from ice. Caliciviruses, influenza A, and some enteroviruses appear to satisfy all three criteria. Environmental ice appears to be an important abiotic reservoir for pathogenic microbes. World health and eradication of specific pathogens could be affected by this huge reservoir.

Caliciviridae↗
PubMed2006

Inflammation and atrial fibrillation: is Chlamydia pneumoniae a candidate pathogen of atrial fibrillation?

Atrial fibrillation is the most common arrhythmia, however, the mechanism of atrial fibrillation is not well explained. It has been considered that inflammation plays a role in atrial fibrillation, recently. Patients undergoing coronary artery bypass graft are at high risk for developing postoperative atrial fibrillation. The peak levels of C-reactive protein (CRP) were paralleled to the incidence of postoperative atrial fibrillation. In general population, CRP was also higher in patients with atrial fibrillation than in control people. Persistent atrial fibrillation patients had a higher CRP level than paroxysmal atrial fibrillation patients. CRP was not only associated with the presence of atrial fibrillation but may also predict patients at increased risk for future development of atrial fibrillation. Why inflammation markers in atrial fibrillation are high is a puzzling problem. We hypothesized that Chlamydia pneumoniae infection is a possible cause of atrial fibrillation by initiating inflammation response. It was demonstrated that infection of endothelial cells with C. pneumoniae elicited the production of Monocyte Chemoattractant Protein-1, interleukin-1, interleukin-8, interleukin-18, tumor necrosis factor, interferon and soluble intercellular adhesion molecule. Most of these cytokines play a crucial role in inflammation response that associate with the initiating and maintenance of atrial fibrillation. There are so many pathogens that can trigger inflammation. Some evidences showed that C. pneumoniae was the most likely pathogen of atrial fibrillation. In epidemic study, the incidence of atrial fibrillation increased from younger to elder and atrial fibrillation was more common in men than in women. C. pneumoniae has the same epidemic trend as the incidence of atrial fibrillation. Hypertension, myocardial infarction and reduced lung function are predictors of atrial fibrillation. C. pneumoniae infection is high in the patients with the above diseases. C. pneumoniae was found in endomyocardial biopsy samples, which supported C. pneumoniae was the candidate pathogen, too. Chlamydia infection can cause myocardial interstitial fibrosis and inflammation cells infiltration. The pathology characters of C. pneumoniae infection are similar to that found in atrial fibrillation. Seroepidemic study should be carried out to evaluate if there is relationship between C. pneumoniae and atrial fibrillation. If the hypothesis is confirmed, macrocyclic lactone antibiotics may be used to eliminate the pathogen. It will be a new target point to treat atrial fibrillation.

Atrial Fibrillation↗
PubMed2004

Contribution of periodontal pathogens on tongue dorsa analyzed with real-time PCR to oral malodor.

Oral malodor is considered to originate primarily from tongue microbiota populations. However, the relationship between oral malodor and tongue microbiota remains unclear. In this study, tongue periodontal pathogens were analyzed via real-time PCR, and the association between oral malodor and tongue periodontal pathogens, including Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, Prevotella nigrescens and Treponema denticola, was examined. The subject population consisted of 29 individuals with and 10 healthy persons without oral malodor. Oral malodor was assessed by organoleptic test and volatile sulfur compound (VSC) levels as measured by gas chromatography. Real-time PCR was conducted for anaerobes in tongue biofilm samples employing a LightCycler system; furthermore, bacterial proportion served as a quantitative parameter. Among the five anaerobes, only T. forsythia displayed higher proportions in malodor subjects than corresponding values in healthy controls. Proportions of P. intermedia and P. nigrescens correlated strongly with hydrogen sulfide concentration. Proportions of P. gingivalis and P. nigrescens also exhibited strong correlation with methyl mercaptan concentration. The correlation coefficient between the proportion of the total of the five anaerobes and total VSC level (r = 0.88) was greater than that between bacterial proportion and organoleptic score (r = 0.29). When a linear regression analysis was performed utilizing the proportion of each of the five periodontal pathogens as an independent variable, the explanatory power of these independent variables revealed 81% for total VSC level and 16% for organoleptic score. These results suggest that these five periodontal pathogens on tongue dorsa may contribute greatly to VSC production.

Adult↗
PubMed2006

Expression profiling of host pathogen interactions: how Mycobacterium tuberculosis and the macrophage adapt to one another.

It has recently become feasible to quantify all mRNAs encoded by the genomes of bacterial pathogens and their eukaryotic host cells and to apply this approach to study the interaction of Mycobacterium tuberculosis with its primary host cell, the macrophage. These studies helped to identify regulatory circuits which mediate adaptation of the M. tuberculosis transcriptome to intraphagosomal environments and stimulated hypotheses for the function of these circuits in human tuberculosis. The macrophage transcriptome reacts to infections with the induction of a pathogen-unspecific expression program as well as the induction of pathogen-specific expression signatures, both of which contribute to the immunologic activation of the infected cell. M. tuberculosis induced changes in the macrophage transcriptome are mediated by Toll-like receptor dependent and Toll-like receptor independent signal transduction pathways. This response is shaped by macrophage produced reactive nitrogen and oxygen molecules and affected by viability and virulence of the pathogen.

Animals↗
PubMed2005

Automated methods for multiplexed pathogen detection.

Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cycler where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides "live vs. dead" capabilities. However, sensitivity of the method will need to be improved for RNA analysis to replace PCR.

Automation↗
PubMed2005

Biofilm formation and dispersal and the transmission of human pathogens.

Several pathogenic bacterial species that are found in the environment can form complex multicellular structures on surfaces known as biofilms. Pseudomonas aeruginosa, Vibrio cholerae and certain species of nontuberculous mycobacteria are examples of human pathogens that form biofilms in natural aquatic environments. We suggest that the dynamics of biofilm formation facilitates the transmission of pathogens by providing a stable protective environment and acting as a nidus for the dissemination of large numbers of microorganisms; both as detached biofilm clumps and by the fluid-driven dispersal of biofilm clusters along surfaces. We also suggest that emerging evidence indicates that biofilm formation conveys a selective advantage to certain pathogens by increasing their ability to persist under diverse environmental conditions.

Bacteria↗
PubMed2006

Licensed to kill: the lifestyle of a necrotrophic plant pathogen.

Necrotrophic plant pathogens have received an increasing amount of attention over the past decade. Initially considered to invade their hosts in a rather unsophisticated manner, necrotrophs are now known to use subtle mechanisms to subdue host plants. The gray mould pathogen Botrytis cinerea is one of the most comprehensively studied necrotrophic fungal plant pathogens. The genome sequences of two strains have been determined. Targeted mutagenesis studies are unraveling the roles played in the infection process by a variety of B. cinerea genes that are required for penetration, host cell killing, plant tissue decomposition or signaling. Our increasing understanding of the tools used by a necrotrophic fungal pathogen to invade plants will be instrumental to designing rational strategies for disease control.

Biomass↗
PubMed2006

Expression of TOLL-like receptors (TLR) by bovine antigen-presenting cells-potential role in pathogen discrimination?

Invading pathogens are controlled by the innate and adaptive arms of the immune system. Adaptive immunity, mediated by B and T lymphocytes, recognises pathogens via high affinity receptors. However, the establishment of a primary adaptive immune response is not rapid enough to eradicate invading microorganisms as it involves cell proliferation, gene activation and protein synthesis. More rapid defence mechanisms are provided by innate immunity, which recognises invading pathogens by germ-line-encoded pattern recognition receptors. Recent evidence shows that this recognition can mainly be attributed to the family of TOLL-like receptors (TLR). Binding of pathogen-associated molecular patterns to TLR induces the production of reactive oxygen and nitrogen intermediates, pro-inflammatory cytokines, and up-regulates expression of co-stimulatory molecules, subsequently initiating the adaptive immunity. In this paper, we will discuss the current knowledge with regards to the TLR, and in particular the bovine family of TLR. In addition, we will show the expression of TLR mRNA in bovine antigen-presenting cell subsets, summarise the discovery and the critical roles of TLR2 in host defence against Mycobacteria, and provide evidence for a mycobacteria species-specific response of bovine macrophages.

Animals↗
PubMed2004

Pathogen carriage by the cat flea Ctenocephalides felis (Bouché) in the United Kingdom.

The carriage of Bartonella, Rickettsia felis and haemoplasma species was investigated in cat fleas (Ctenocephalides felis) collected from 121 cats and dogs in the United Kingdom. DNA extracted from fleas was analysed using genus and species-specific PCR and amplicons were characterised using DNA sequencing. Fifty percent of flea samples were PCR positive for at least one pathogen. Twenty one percent were positive for R. felis, 17% for Bartonella henselae, 40% for haemoplasma species and 20% were infected with more than one of the pathogen species studied. It is clear from the results in this study that companion cats and dogs are commonly infested with Ct. felis carrying bacterial pathogens of significance to human and animal health. These findings raise the possibility that Ct. felis found on dogs and cats are a potential source of infection with such pathogens for humans.

Animals↗
PubMed2005

Scrambling of the amino acids within the transmembrane domain of Vpu results in a simian-human immunodeficiency virus (SHIVTM) that is less pathogenic for pig-tailed macaques.

Previous studies have shown that the transmembrane (TM) domain of the subtype B Vpu enhances virion release from cells and some studies have shown that this domain may form an oligomeric structure with properties of an ion channel. To date, no studies have been performed to assess the role of this domain in virus pathogenesis in a macaque model of disease. Using a pathogenic molecular clone of simian human immunodeficiency virus (SHIVKU-1bMC33), we have generated a novel virus in which the transmembrane domain of the Vpu protein was scrambled but maintained hydrophobic in nature (SHIVTM), which presumably would disrupt any ion channel TM properties of this protein. Vectors expressing the Vpu as a fusion protein with the enhanced green fluorescent protein (VpuTMEGFP) indicate that it was transported to the same intracellular compartment as the unmodified Vpu protein but did not down-regulate cell surface expression of CD4. To assess the pathogenicity of SHIVTM, three pig-tailed macaques were inoculated with the SHIVTM and monitored for 6-8 months for CD4+ T cell levels, viral loads and the stability of the sequence of the vpu gene. Our results indicated that unlike the parental SHIVKU-1bMC33, inoculation of macaques with SHIVTM did not cause a severe CD4+ T cell loss over the course of their infections. Sequence analysis of the vpu gene analyzed from sequential PBMC samples derived from macaques revealed that the scrambled TM was stable during the course of infection. At necropsy, examination of tissues revealed low viral loads and none of the pathology commonly observed in lymphoid and non-lymphoid tissues following inoculation with the pathogenic parental SHIVKU-1bMC33 virus. Thus, these results show for the first time that the TM domain of Vpu contributes to the pathogenicity of SHIVKU-1bMC33 in pig-tailed macaques.

Acquired Immunodeficiency Syndrome↗
PubMed2005

Multiple amino acids in the glycoprotein of rabies virus are responsible for pathogenicity in adult mice.

We have reported that the region between amino acids 164 and 303 in the glycoprotein of rabies Nishigahara strain is important for lethality in adult mice. The region contains nine amino acid substitutions between the virulent Nishigahara and the avirulent RC-HL strains. In order to determine key residues for the pathogenicity, two chimeric strains and seven mutants were generated and examined for pathogenicities. The R(G 242/255/268) strain, in which amino acids at positions 242, 255, and 268 were replaced by those from the Nishigahara strain in the genomic backbone of the RC-HL strain, showed the same lethality as that of the Nishigahara strain in mice. Mutants in which one or two of these three amino acids were replaced by those from the Nishigahara strain did not revert to the lethality of the R(G 242/255/268) strain. These results demonstrate that at least these three amino acids are related to enhancement of pathogenicity. It is thought that multiple amino acids of the G protein are related to the pathogenicity of rabies viruses.

Amino Acid Substitution↗
PubMed2005

Factors controlling pathogen destruction during anaerobic digestion of biowastes.

Anaerobic digestion is the principal method of stabilising biosolids from urban wastewater treatment in the UK, and it also has application for the treatment of other types of biowaste. Increasing awareness of the potential risks to human and animal health from environmental sources of pathogens has focused attention on the efficacy of waste treatment processes at destroying pathogenic microorganisms in biowastes recycled to agricultural land. The degree of disinfection achieved by a particular anaerobic digester is influenced by a variety of interacting operational variables and conditions, which can often deviate from the ideal. Experimental investigations demonstrate that Escherichia coli and Salmonella spp. are not damaged by mesophilic temperatures, whereas rapid inactivation occurs by thermophilic digestion. A hydraulic, biokinetic and thermodynamic model of pathogen inactivation during anaerobic digestion showed that a 2 log10 reduction in E. coli (the minimum removal required for agricultural use of conventionally treated biosolids) is likely to challenge most conventional mesophilic digesters, unless strict maintenance and management practices are adopted to minimise dead zones and by-pass flow. Efficient mixing and organic matter stabilisation are the main factors controlling the rate of inactivation under mesophilic conditions and not a direct effect of temperature per se on pathogenic organisms.

Bacteria, Anaerobic↗