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Influence of taxonomic status on the in vitro antimicrobial susceptibility of the Burkholderia cepacia complex.

The Burkholderia cepacia complex is a diverse group of human pathogens that cause life-threatening lung infections in patients with cystic fibrosis or chronic granulomatous disease, and in patients requiring intensive care. Most previous antimicrobial susceptibility studies of these bacteria were performed before recent major revisions in the taxonomy of these bacteria. We determined the in vitro susceptibility of 65 B. cepacia complex isolates from clinical and environmental sources, representing six genomovars. Although intrinsic resistance is considered to be a common feature of the B. cepacia complex, MICs of individual antimicrobials varied widely and resistance was not exhibited by all members of the group.

Burkholderia cepacia↗

Identification and masking of artifactual and misleading within-host variants in deep-sequencing SARS-CoV-2 data.

Deep-sequencing data are increasingly used to study within-host viral diversity and to inform evolutionary inference. For SARS-CoV-2, analyses based on intra-host single-nucleotide variants (iSNVs) have been widely applied to quantify within-host diversity and infer transmission dynamics. However, these applications critically depend on the reliable identification of low-frequency variants, which remain vulnerable to systematic and technical artifacts. In this study, we show that recurrent artifactual iSNVs are common in large-scale SARS-CoV-2 sequencing data and can persist even under conservative minor allele frequency thresholds. Using data from the UK's Office for National Statistics COVID-19 Infection Survey, we demonstrate that such artifacts are predominantly sequencing center-specific rather than primer-specific. Each center exhibits a modest, distinct set of recurrent artifactual variants showing little overlap with sites routinely masked at the consensus level. To address this, we developed a systematic, dataset-aware framework that uses recurrence within sequencing datasets to identify small, noise-adapted sets of artifactual iSNVs to mask. Applying this framework reduces spurious sharing of low-frequency variants between samples and qualitatively alters downstream inferences, including estimates of within-host diversity and transmission bottleneck sizes. Although this study focused on SARS-CoV-2, it is likely that recurrent artifactual iSNVs will be problematic for other viruses as mass-sequencing becomes increasingly routine. Together, these findings highlight the importance of explicit, dataset-aware artifact control for robust inference from within-host variation, particularly as genomic studies increasingly seek to exploit sub-consensus diversity in rapidly evolving pathogens.

Humans↗

Natural regulators of T-cell lymphokine production in vivo.

The mammalian immune system possesses the intrinsic capacity to evoke a wide variety of functionally distinct effector mechanisms following stimulation by a particular antigenic substance. Such diversity in available responses is absolutely essential to the immunocompetent host, which must continually deal with a diverse set of potential pathogens within its ever-changing environment. The development of appropriate types of immune responses, therefore, represents a highly dynamic process that requires that an equivalent consideration be given to a large array of components, any one of which is capable of modulating the final outcome. While the nature and complexity of the antigen(s), plus the intracellular or extracellular mode of presentation, provide specificity and some selection to the developing process, the route of antigen entry, as well as the physiological status of the host at the time of antigen insult, also contribute significantly to the formation of any immune response. The overall objective of this article is to introduce the concept that platelet-derived growth factor (PDGF) (either preformed or synthesized in response to stimulation), plus a number of steroid hormones (some of which are end-organ metabolized at local tissue sites), can all play significant roles in the genesis of immunologic responses in vivo.

Aging↗

Interference competition and parasite virulence.

Within-host competition between parasites, a consequence of infection by multiple strains, is predicted to favour rapid host exploitation and greater damage to hosts (virulence). However, the inclusion of biological variables can drastically change this relationship. For example, if competing parasite strains produce toxins that kill each other (interference competition), their growth rates and virulence may be reduced relative to single-strain infections. Bacteriocins are antimicrobial toxins produced by bacteria that target closely related strains and species, and to which the producing strain is immune. We investigated competition between bacteriocin-producing, insect-killing bacteria (Photorhabdus and Xenorhabdus) and how this competition affected virulence in caterpillars. Where one strain could kill the other, and not vice versa, the non-killing strain was competitively excluded, and insect mortality was the same as that of the killing strain alone. However, when caterpillars were multiply infected by strains that could kill each other, we did not observe competitive exclusion and their virulence was less than single-strain infections. The ubiquity and diversity of bacteriocins among pathogenic bacteria suggest mixed infections will be, on average, less virulent than single infections.

Animals↗

Active use of the metapleural glands by ants in controlling fungal infection.

Insect societies face constant challenges from disease agents. Ants deploy diverse antimicrobial compounds against pathogens and the key sources are metapleural glands (MGs). Are MG products passively secreted and used indiscriminately or are they selectively used when ants are challenged by pathogens? In 26 species from five subfamilies, ants use foreleg movements to precisely groom the MG opening. In the absence of experimental infection, MG grooming rates are low and workers groom themselves after contacting the MGs. The derived leaf-cutter ants (Atta and Acromyrmex) also groom their fungal gardens, substrata (leaves), queens and nest-mates after MG grooming. Atta respond to a challenge by fungal conidia by increasing the rate of MG grooming, but do not do so when an inert powder is applied. This increase occurs in the first hour after a potential infection, after which it returns to baseline levels. Ants with open MGs produce more infrabuccal pellets (IP) than ants with sealed MGs and conidia within pellets from the former are less likely to germinate. Thus, ants selectively groom their MGs when disease agents are present, suggesting that they also selectively use their MG secretions, which has important implications for understanding the evolution of hygienic behaviour in social groups.

Animals↗

Heterogeneity within the gram-positive anaerobic cocci demonstrated by analysis of 16S-23S intergenic ribosomal RNA polymorphisms.

Peptostreptococci are gram-positive, strictly anaerobic bacteria which, although regarded as members of the commensal human microflora, are also frequently isolated from sites of clinical infection. The study of this diverse group of opportunist pathogens has been hindered by an inadequate taxonomy and the lack of a valid identification scheme. Recent re-classification of the Peptostreptococcus family into five distinct genus groups has helped to clarify the situation. However, this has been on the basis of 16S rRNA sequence determinations, which are both time-consuming and expensive. The aim of the present study was to evaluate the use of PCR-amplified ribosomal DNA spacer polymorphisms for the rapid differentiation of the currently recognised taxa within the group of anaerobic gram-positive cocci. A collection comprising 19 reference strains with representatives of each of the 15 species, two close relatives and two of the well-characterised groups, together with 38 test strains was studied. All strains were identified to species group level by phenotypic means. Amplification of the 16S-23S intergenic spacer region (ISR) with universal primers produced distinct banding patterns for all the 19 reference strains and the patterns could be differentiated easily visually. However, of the 38 test strains, less than half could be speciated from ISR analysis alone. Only five groups produced correlating banding patterns for all members tested (Peptoniphilus lacrimalis, P. ivorii, Anaerococcus octavius, Peptostreptococcus anaerobius and Micromonas micros). For other species, either the type strain differed significantly from other species members (e.g., A. hydrogenalis) or there appeared to be considerable intra-species variation (e.g., A. vaginalis). Partial 16S rRNA gene sequences for the 'trisimilis' and 'betaGAL' groups showed that both are most closely related to the Anaerococcus group. This work highlights the heterogeneous nature of a number of Peptostreptococcus species and hence the need for still further revision of the taxonomy of this important group of pathogens.

Anaerobiosis↗

EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. I. Evidence for stage-specific gene expression in aerial conidia, in vitro blastospores and submerged conidia.

The entomopathogenic fungus Beauveria (Cordyceps) bassiana holds much promise as a pest biological control agent. B. bassiana produces at least three in vitro single cell infectious propagules, including aerial conidia, vegetative cells termed blastospores and submerged conidia, that display different morphological, biochemical and virulence properties. Populations of aerial conidia, blastospores and submerged conidia were produced on agar plates, rich liquid broth cultures and under conditions of nutrient limitation in submerged cultures, respectively. cDNA libraries were generated from mRNA isolated from each B. bassiana cell type and approximately 2,500 5' end sequences were determined from each library. Sequences derived from aerial conidia clustered into 284 contigs and 963 singlets, with those derived from blastospores and submerged conidia forming 327 contigs with 788 singlets, and 303 contigs and 1,079 contigs, respectively. Almost half (40-45 %) of the sequences in each library displayed either no significant similarity (e value >10(-4)) or similarity to hypothetical proteins found in the NCBI database. The expressed sequence tag dataset also included sequences representing a significant portion of proteins in cellular metabolism, information storage and processing, transport and cell processes, including cell division and posttranslational modifications. Transcripts encoding a diverse array of pathogenicity-related genes, including proteases, lipases, esterases, phosphatases and enzymes producing toxic secondary metabolites, were also identified. Comparative analysis between the libraries identified 2,416 unique sequences, of which 20-30 % were unique to each library, and only approximately 6 % of the sequences were shared between all three libraries. The unique and divergent representation of the B. bassiana transcriptome in the cDNA libraries from each cell type suggests robust differential gene expression profiles in response to environmental conditions.

Animals↗

Detection of Magnaporthe grisea in infested rice seeds using polymerase chain reaction.

AIM: To develop a diagnostic assay based on polymerase chain reaction for the detection of Magnaporthe grisea from infested rice seeds. METHODS AND RESULTS: Primers were designed based on the nucleotide sequence of the mif 23, an infection-specific gene of M. grisea. The primers amplified target DNA from genetically and geographically diverse isolates of the pathogen. The lowest concentration of template DNA that led to amplification was 20 rhog. No PCR product was detected when DNA from other fungi was used, indicating the specificity of the primers. With this PCR based seed assay, M. grisea was detected in rice seedlots with infestation rates as low as 0.2%. CONCLUSION: The PCR detection of M. grisea is simple, rapid, specific, sensitive and suitable for the routine detection of the pathogen in infested seeds. SIGNIFICANCE AND IMPACT OF THE STUDY: Introduction of the blast fungus into new areas where it has not been previously recorded could be avoided by the detection of infested seedlots. A PCR-based seed assay could facilitate risk assessment of naturally infested rice seeds; help design management programs and optimize fungicide use.

DNA, Fungal↗

Protein misfolding in neurodegenerative diseases.

A common pathogenic mechanism shared by diverse neurodegenerative disorders, like Alzheimer's disease, Parkinson's disease, Huntington's disease and transmissible spongiform encephalopathies, may be altered protein homeostasis leading to protein misfolding and aggregation of a wide variety of different proteins in the form of insoluble fibrils. Mutations in the genes encoding protein constituents of these aggregates have been linked to the corresponding diseases, thus a reasonable scenario of pathogenesis was based on misfolding of a neurone-specific protein that forms insoluble fibrils that subsequently kill neuronal cells. However, during the past 5 years accumulating evidence has revealed the neurotoxic role of prefibrillar intermediate forms (soluble oligomers and protofibrils) produced during fibril formation. Many think these may be the predominant neurotoxic species, whereas microscopically visible fibrillar aggregates may not be toxic. Large protein aggregates may rather be simply inactive, or even represent a protective state that sequesters and inactivates toxic oligomers and protofibrils. Further understanding of the biochemical mechanisms involved in protein misfolding and fibrillization may optimize the planning of common therapeutic approaches for neurodegenerative diseases, directed towards reversal of protein misfolding, blockade of protein oligomerization and interference with the action of toxic proteins.

Alzheimer Disease↗

Bacterial contamination of platelet concentrates prepared by different methods: results of standardized sterility testing in Germany.

BACKGROUND AND OBJECTIVES: National guidelines for monitoring the bacterial contamination rate of blood components were introduced in Germany in 1997. The objective of this study was to present and evaluate the results of sterility testing of platelet concentrates (PCs) prepared by different methods. MATERIALS AND METHODS: The analysis of results of sterility testing of blood component production from transfusion medicine centres in Germany in 1998 and 2001 was based on information collected using standardized questionnaires. RESULTS: The bacterial contamination rates for single-donor PCs derived from whole blood and apheresis (0.210% vs. 0.156%) were comparable and showed no significant difference. However, pooled PCs produced from four buffy coats using the sterile docking procedure showed a significantly higher bacterial contamination rate compared with single-donor PCs derived from whole blood and apheresis (0.184% vs. 0.604%). CONCLUSIONS: Use of standardized methods for sterility monitoring is sufficient to assess collection and production processes in terms of hygiene and yields reliable data on bacterial contamination rates of blood components. The methods described are suitable for using to analyse the efficiency of newly introduced methods to reduce bacterial contamination rates of blood components (e.g. diversion, bacteria screening and pathogen inactivation).

Bacterial Infections↗

Resistance of fluorescent-labelled Actinobacillus actinomycetemcomitans strains to phagocytosis and killing by human neutrophils.

Neutrophils are initially the predominant cells involved in the host defence of bacterial infections, including periodontal disease. Aggressive periodontitis is associated with Actinobacillus actinomycetemcomitans, a Gram-negative capnophilic microorganism. Infections caused by A. actinomycetemcomitans are not resolved by the host immune response despite the accumulation of neutrophils at the site of inflammation. To better understand the role of natural host defence mechanisms in A. actinomycetemcomitans infections, the interaction of phenotypically diverse strains of this pathogen with human neutrophils was assessed directly using techniques such as genetic labelling with the gene for green fluorescent protein, fluorescence-activated cell sorting and fluorescence imaging. The study included clinical isolates of A. actinomycetemcomitans represented by self-aggregating, biofilm-associated and isogenic planktonic variants. Data obtained showed that complement-mediated phagocytosis of A. actinomycetemcomitans was generally inefficient regardless of strain-specific serotype or leukotoxin production. Furthermore, the majority of ingested bacteria remained viable after exposure to neutrophils for 1 h. Interestingly, uptake of antibody-opsonized bacteria resulted in the rapid cell death of neutrophils. This was in contrast to ingestion of complement-opsonized bacteria, which did not affect neutrophil viability. The methods used in this study provided reliable and reproducible results with respect to adherence, phagocytosis and killing of A. actinomycetemcomitans when encountering human neutrophils.

Aggregatibacter actinomycetemcomitans↗

Recovery of new integron classes from environmental DNA.

Integrons are genetic elements known for their role in the acquisition and expression of genes conferring antibiotic resistance. Such acquisition is mediated by an integron-encoded integrase, which captures genes that are part of gene cassettes. To test whether integrons occur in environments with no known history of antibiotic exposure, PCR primers were designed to conserved regions of the integrase gene and the gene cassette recombination site. Amplicons generated from four environmental DNA samples contained features typical of the integrons found in antibiotic-resistant and pathogenic bacteria. The sequence diversity of the integrase genes in these clones was sufficient to classify them within three new classes of integron. Since they are derived from environments not associated with antibiotic use, integrons appear to be more prevalent in bacteria than previously observed.

Amino Acid Sequence↗

Dynamic nuclear actin assembly by Arp2/3 complex and a baculovirus WASP-like protein.

Diverse bacterial and viral pathogens induce actin polymerization in the cytoplasm of host cells to facilitate infection. Here, we describe a pathogenic mechanism for promoting dynamic actin assembly in the nucleus to enable viral replication. The baculovirus Autographa californica multiple nucleopolyhedrovirus induced nuclear actin polymerization by translocating the host actin-nucleating Arp2/3 complex into the nucleus, where it was activated by p78/83, a viral Wiskott-Aldrich syndrome protein (WASP)-like protein. Nuclear actin assembly by p78/83 and Arp2/3 complex was essential for viral progeny production. Recompartmentalizing dynamic host actin may represent a conserved mode of pathogenesis and reflect viral manipulation of normal functions of nuclear actin.

Actin-Related Protein 2-3 Complex↗

Seasonal abundance of total and pathogenic Vibrio parahaemolyticus in Alabama oysters.

Recent Vibrio parahaemolyticus outbreaks associated with consumption of raw shellfish in the United States focused attention on the occurrence of this organism in shellfish. From March 1999 through September 2000, paired oyster samples were collected biweekly from two shellfish-growing areas in Mobile Bay, Ala. The presence and densities of V. parahaemolyticus were determined by using DNA probes targeting the thermolabile hemolysin (tlh) and thermostable direct hemolysin (tdh) genes for confirmation of total and pathogenic V. parahaemolyticus, respectively. V. parahaemolyticus was detected in all samples with densities ranging from <10 to 12,000 g(-1). Higher V. parahaemolyticus densities were associated with higher water temperatures. Pathogenic strains were detected in 34 (21.8%) of 156 samples by direct plating or enrichment. Forty-six of 6,018 and 31 of 6,992 V. parahaemolyticus isolates from enrichments and direct plates, respectively, hybridized with the tdh probe. There was an apparent inverse relationship between water temperature and the prevalence of pathogenic strains. Pathogenic strains were of diverse serotypes, and 97% produced urease and possessed a tdh-related hemolysin (trh) gene. The O3:K6 serotype associated with pandemic spread and recent outbreaks in the United States was not detected. The efficient screening of numerous isolates by colony lift and DNA probe procedures may account for the higher prevalence of samples with tdh(+) V. parahaemolyticus than previously reported.

Alabama↗

A multigene phylogenetic study of clonal diversity and divergence in North American strains of the plant pathogen Xylella fastidiosa.

Xylella fastidiosa is a pathogen that causes leaf scorch and related diseases in over 100 plant species, including Pierce's disease in grapevines (PD), phony peach disease (PP), plum leaf scald (PLS), and leaf scorch in almond (ALS), oak (OAK), and oleander (OLS). We used a high-resolution DNA sequence approach to investigate the evolutionary relationships, geographic variation, and divergence times among the X. fastidiosa isolates causing these diseases in North America. Using a large data set of 10 coding loci and 26 isolates, the phylogeny of X. fastidiosa defined three major clades. Two of these clades correspond to the recently identified X. fastidiosa subspecies piercei (PD and some ALS isolates) and X. fastidiosa subsp. multiplex (OAK, PP, PLS, and some ALS isolates). The third clade grouped all of the OLS isolates into a genetically distinct group, named X. fastidiosa subsp. sandyi. These well-differentiated clades indicate that, historically, X. fastidiosa has been a clonal organism. Based on their synonymous-site divergence ( approximately 3%), these three clades probably originated more than 15,000 years ago, long before the introduction of the nonnative plants that characterize most infections. The sister clades of X. fastidiosa subsp. sandyi and X. fastidiosa subsp. piercei have synonymous-site evolutionary rates 2.9 times faster than X. fastidiosa subsp. multiplex, possibly due to generation time differences. Within X. fastidiosa subsp. multiplex, a low level ( approximately 0.1%) of genetic differentiation indicates the recent divergence of ALS isolates from the PP, PLS, and OAK isolates due to host plant adaptation and/or allopatry. The low level of variation within the X. fastidiosa subsp. piercei and X. fastidiosa subsp. sandyi clades, despite their antiquity, suggests strong selection, possibly driven by host plant adaptation.

Bacterial Proteins↗

Blastocystis ratti induces contact-independent apoptosis, F-actin rearrangement, and barrier function disruption in IEC-6 cells.

Blastocystis is an enteric protozoan purportedly associated with numerous clinical cases of diarrhea, flatulence, vomiting, and other gastrointestinal symptoms. Despite new knowledge of Blastocystis cell biology, genetic diversity, and epidemiology, its pathogenic potential remains controversial. Numerous clinical and epidemiological studies either implicate or exonerate the parasite as a cause of intestinal disease. Therefore, the aim of this study was to investigate the pathogenic potential of Blastocystis by studying the interactions of Blastocystis ratti WR1, an isolate of zoonotic potential, with a nontransformed rat intestinal epithelial cell line, IEC-6. Here, we report that B. ratti WR1 induces apoptosis in IEC-6 cells in a contact-independent manner. Furthermore, we found that B. ratti WR1 rearranges F-actin distribution, decreases transepithelial resistance, and increases epithelial permeability in IEC-6 cell monolayers. In addition, we found that the effects of B. ratti on transepithelial electrical resistance and epithelial permeability were significantly abrogated by treatment with metronidazole, an antiprotozoal drug. Our results suggest for the first time that Blastocystis-induced apoptosis in host cells and altered epithelial barrier function might play an important role in the pathogenesis of Blastocystis infections and that metronidazole has therapeutic potential in alleviating symptoms associated with Blastocystis.

Actins↗

Decreased infectivity in Borrelia burgdorferi strain B31 is associated with loss of linear plasmid 25 or 28-1.

Previous reports indicated a correlation between loss of plasmids and decreased infectivity of Borrelia burgdorferi strain B31, suggesting that plasmids may encode proteins that are required for pathogenesis. In this study, we expand on this correlation. Using the B. burgdorferi genomic sequence, we designed primers specific for each plasmid, and by using PCR we catalogued 11 linear and 2 circular plasmids from 49 clonal isolates of a mid-passage B. burgdorferi strain B31, initially derived from infected mouse skin, and 20 clones obtained from mouse skin infected with a low-passage isolate of B. burgdorferi strain B31. Among the 69 clones analyzed, nine distinct genotypes were identified relative to wild-type B. burgdorferi strain B31. Among the nine clonal genotypes obtained, only the 9-kb circular plasmid (cp9), the 25-kb linear plasmid (lp25), and either the 28-kb linear plasmid 1 or 4 (lp28-1 and lp28-4, respectively) were missing, in different combinations. We compared the infectivity of the wild-type strain, containing all known B. burgdorferi plasmids, with those of single mutants lacking either lp28-1, lp28-4, or lp25 and a double mutant missing both cp9 and lp28-1. The infectivity data indicated that B. burgdorferi strain B31 cells lacking lp28-4 were modestly attenuated in all tissues analyzed, whereas samples missing lp25 were completely attenuated in all tissues, even at the highest inoculum tested. Isolates without lp28-1 infected the joint tissue yet were not able to infect other tissues as effectively. In addition, we have observed a selection in vivo in the skin, bladder, and joint for cells containing lp25 and in the skin and bladder for cells containing lp28-1, indicating that lp25 and lp28-1 encode proteins required for colonization and short-term maintenance in these mammalian tissues. In contrast, there was no selection in the joint for cells containing lp28-1, suggesting that genes on lp28-1 are not required for colonization of B. burgdorferi within the joint. These observations imply that the dynamic nature of the B. burgdorferi genome may provide the genetic heterogeneity necessary for survival in the diverse milieus that this pathogen occupies in nature and may contribute to tropism in certain mammalian host tissues.

Animals↗

Bacteriophage HP2 of Haemophilus influenzae.

Temperate bacteriophages effect chromosomal evolution of their bacterial hosts, mediating rearrangements and the acquisition of novel genes from other taxa. Although the Haemophilus influenzae genome shows evidence of past phage-mediated lateral transfer, the phages presumed responsible have not been identified. To date, six different H. influenzae phages are known; of these, only the HP1/S2 group, which lyosogenizes exclusively Rd strains (which were originally encapsulated serotype d), is well characterized. Phages in this group are genetically very similar, with a highly conserved set of genes. Because the majority of H. influenzae strains are nonencapsulated (nontypeable), it is important to characterize phages infecting this larger, genetically more diverse group of respiratory pathogens. We have identified and sequenced HP2, a bacteriophage of nontypeable H. influenzae. Although related to the fully sequenced HP1 (and even more so to the partially sequenced S2) and similar in genetic organization, HP2 has a few novel genes and differs in host range; HP2 will not infect or lysogenize Rd strains. Genomic comparisons between HP1/S2 and HP2 suggest recent divergence, with new genes completely replacing old ones at certain loci. Sequence comparisons suggest that H. influenzae phages evolve by recombinational exchange of genes with each other, with cryptic prophages, and with the host chromosome.

Amino Acid Sequence↗