Do standard burn mortality formulae work on a population of severely burned children and adults?

Accurate prediction of mortality following burns is useful as an audit tool, and for providing treatment plan and resource allocation criteria. Common burn formulae (Ryan Score, Abbreviated Burn Severity Index (ABSI), classic and revised Baux) have not been compared with the standard Acute Physiology and Chronic Health Evaluation II (APACHEII) or re-validated in a severely (≥20% total burn surface area) burned population. Furthermore, the revised Baux (R-Baux) has been externally validated thoroughly only once and the pediatric Baux (P-Baux) has yet to be. Using 522 severely burned patients, we show that burn formulae (ABSI, Baux, revised Baux) outperform APACHEII among adults (AUROC increase p<0.001 adults; p>0.5 children). The Ryan Score performs well especially among the most at-risk populations (estimated mortality [90% CI] original versus current study: 33% [26-41%] versus 30.18% [24.25-36.86%] for Ryan Score 2; 87% [78-93%] versus 66.48% [51.31-78.87%] for Ryan Score 3). The R-Baux shows accurate discrimination (AUROC 0.908 [0.869-0.947]) and is well-calibrated. However, the ABSI and P-Baux, although showing high measures of discrimination (AUROC 0.826 [0.737-0.916] and 0.848 [0.758-0.938]) in children), exceedingly overestimates mortality, indicating poor calibration. We highlight challenges in designing and employing scores that are applicable to a wide range of populations.

Prediction of multiple infections after severe burn trauma: a prospective cohort study.

OBJECTIVE: To develop predictive models for early triage of burn patients based on hypersusceptibility to repeated infections. BACKGROUND: Infection remains a major cause of mortality and morbidity after severe trauma, demanding new strategies to combat infections. Models for infection prediction are lacking. METHODS: Secondary analysis of 459 burn patients (≥16 years old) with 20% or more total body surface area burns recruited from 6 US burn centers. We compared blood transcriptomes with a 180-hour cutoff on the injury-to-transcriptome interval of 47 patients (≤1 infection episode) to those of 66 hypersusceptible patients [multiple (≥2) infection episodes (MIE)]. We used LASSO regression to select biomarkers and multivariate logistic regression to built models, accuracy of which were assessed by area under receiver operating characteristic curve (AUROC) and cross-validation. RESULTS: Three predictive models were developed using covariates of (1) clinical characteristics; (2) expression profiles of 14 genomic probes; (3) combining (1) and (2). The genomic and clinical models were highly predictive of MIE status [AUROCGenomic = 0.946 (95% CI: 0.906-0.986); AUROCClinical = 0.864 (CI: 0.794-0.933); AUROCGenomic/AUROCClinical P = 0.044]. Combined model has an increased AUROCCombined of 0.967 (CI: 0.940-0.993) compared with the individual models (AUROCCombined/AUROCClinical P = 0.0069). Hypersusceptible patients show early alterations in immune-related signaling pathways, epigenetic modulation, and chromatin remodeling. CONCLUSIONS: Early triage of burn patients more susceptible to infections can be made using clinical characteristics and/or genomic signatures. Genomic signature suggests new insights into the pathophysiology of hypersusceptibility to infection may lead to novel potential therapeutic or prophylactic targets.

Alternatives to vitamin B1 uptake revealed with discovery of riboswitches in multiple marine eukaryotic lineages.

Vitamin B1 (thiamine pyrophosphate, TPP) is essential to all life but scarce in ocean surface waters. In many bacteria and a few eukaryotic groups thiamine biosynthesis genes are controlled by metabolite-sensing mRNA-based gene regulators known as riboswitches. Using available genome sequences and transcriptomes generated from ecologically important marine phytoplankton, we identified 31 new eukaryotic riboswitches. These were found in alveolate, cryptophyte, haptophyte and rhizarian phytoplankton as well as taxa from two lineages previously known to have riboswitches (green algae and stramenopiles). The predicted secondary structures bear hallmarks of TPP-sensing riboswitches. Surprisingly, most of the identified riboswitches are affiliated with genes of unknown function, rather than characterized thiamine biosynthesis genes. Using qPCR and growth experiments involving two prasinophyte algae, we show that expression of these genes increases significantly under vitamin B1-deplete conditions relative to controls. Pathway analyses show that several algae harboring the uncharacterized genes lack one or more enzymes in the known TPP biosynthesis pathway. We demonstrate that one such alga, the major primary producer Emiliania huxleyi, grows on 4-amino-5-hydroxymethyl-2-methylpyrimidine (a thiamine precursor moiety) alone, although long thought dependent on exogenous sources of thiamine. Thus, overall, we have identified riboswitches in major eukaryotic lineages not known to undergo this form of gene regulation. In these phytoplankton groups, riboswitches are often affiliated with widespread thiamine-responsive genes with as yet uncertain roles in TPP pathways. Further, taxa with 'incomplete' TPP biosynthesis pathways do not necessarily require exogenous vitamin B1, making vitamin control of phytoplankton blooms more complex than the current paradigm suggests.

Comparative genomics of multiple strains of Pseudomonas cannabina pv. alisalensis, a potential model pathogen of both monocots and dicots.

Comparative genomics of closely related pathogens that differ in host range can provide insights into mechanisms of host-pathogen interactions and host adaptation. Furthermore, sequencing of multiple strains with the same host range reveals information concerning pathogen diversity and the molecular basis of virulence. Here we present a comparative analysis of draft genome sequences for four strains of Pseudomonas cannabina pathovar alisalensis (Pcal), which is pathogenic on a range of monocotyledonous and dicotyledonous plants. These draft genome sequences provide a foundation for understanding host range evolution across the monocot-dicot divide. Like other phytopathogenic pseudomonads, Pcal strains harboured a hrp/hrc gene cluster that codes for a type III secretion system. Phylogenetic analysis based on the hrp/hrc cluster genes/proteins, suggests localized recombination and functional divergence within the hrp/hrc cluster. Despite significant conservation of overall genetic content across Pcal genomes, comparison of type III effector repertoires reinforced previous molecular data suggesting the existence of two distinct lineages within this pathovar. Furthermore, all Pcal strains analyzed harbored two distinct genomic islands predicted to code for type VI secretion systems (T6SSs). While one of these systems was orthologous to known P. syringae T6SSs, the other more closely resembled a T6SS found within P. aeruginosa. In summary, our study provides a foundation to unravel Pcal adaptation to both monocot and dicot hosts and provides genetic insights into the mechanisms underlying pathogenicity.

The plant pathogen Pseudomonas syringae pv. tomato is genetically monomorphic and under strong selection to evade tomato immunity.

Recently, genome sequencing of many isolates of genetically monomorphic bacterial human pathogens has given new insights into pathogen microevolution and phylogeography. Here, we report a genome-based micro-evolutionary study of a bacterial plant pathogen, Pseudomonas syringae pv. tomato. Only 267 mutations were identified between five sequenced isolates in 3,543,009 nt of analyzed genome sequence, which suggests a recent evolutionary origin of this pathogen. Further analysis with genome-derived markers of 89 world-wide isolates showed that several genotypes exist in North America and in Europe indicating frequent pathogen movement between these world regions. Genome-derived markers and molecular analyses of key pathogen loci important for virulence and motility both suggest ongoing adaptation to the tomato host. A mutational hotspot was found in the type III-secreted effector gene hopM1. These mutations abolish the cell death triggering activity of the full-length protein indicating strong selection for loss of function of this effector, which was previously considered a virulence factor. Two non-synonymous mutations in the flagellin-encoding gene fliC allowed identifying a new microbe associated molecular pattern (MAMP) in a region distinct from the known MAMP flg22. Interestingly, the ancestral allele of this MAMP induces a stronger tomato immune response than the derived alleles. The ancestral allele has largely disappeared from today's Pto populations suggesting that flagellin-triggered immunity limits pathogen fitness even in highly virulent pathogens. An additional non-synonymous mutation was identified in flg22 in South American isolates. Therefore, MAMPs are more variable than expected differing even between otherwise almost identical isolates of the same pathogen strain.

Reconstructing host range evolution of bacterial plant pathogens using Pseudomonas syringae pv. tomato and its close relatives as a model.

Several lines of evidence suggest that highly virulent bacterial human pathogens evolved from less virulent wider host range animal pathogens since human migration out of Africa. To investigate evolution of host specificity of bacterial plant pathogens, here we report a molecular evolutionary analysis of the model plant pathogen Pseudomonas syringae pv. tomato DC3000 and of close relatives that are pathogens of a diverse set of crop plants. Extensive host range tests on five different plant species were performed. Combining phylogenetic data with host range data, a reconstruction of host range of all putative ancestors was performed. In particular, the hypothesis was tested that highly virulent narrow host range pathogens of today's crops grown in monoculture evolved from ancestors with wider host range that were adapted to natural mixed plant communities of pre-agricultural times. We found support for this hypothesis in individual clades. However, reconstruction of host range of the most recent common ancestor of all analyzed strains was not conclusive. Based on the obtained results we stress the importance of including pathogens from wild plants when reconstructing the evolution of plant pathogenic bacteria.

PAMDB, a multilocus sequence typing and analysis database and website for plant-associated microbes.

Although there are adequate DNA sequence differences among plant-associated and plant-pathogenic bacteria to facilitate molecular approaches for their identification, identification at a taxonomic level that is predictive of their phenotype is a challenge. The problem is the absence of a taxonomy that describes genetic variation at a biologically relevant resolution and of a database containing reference strains for comparison. Moreover, molecular evolution, population genetics, ecology, and epidemiology of many plant-pathogenic and plant-associated bacteria are still poorly understood. To address these challenges, a database with web interface was specifically designed for plant-associated and plant-pathogenic microorganisms. The Plant-Associated Microbes Database (PAMDB) comprises, thus far, data from multilocus sequence typing and analysis (MLST/MLSA) studies of Acidovorax citrulli, Pseudomonas syringae, Ralstonia solanacearum, and Xanthomonas spp. Using data deposited in PAMDB, a robust phylogeny of Xanthomonas axonopodis and related bacteria has been inferred, and the diversity existing in the Xanthomonas genus and in described Xanthomonas spp. has been compared with the diversity in P. syringae and R. solanacearum. Moreover, we show how PAMDB makes it easy to distinguish between different pathogens that cause almost identical diseases. The scalable design of PAMDB will make it easy to add more plant pathogens in the future.

A draft genome sequence of Pseudomonas syringae pv. tomato T1 reveals a type III effector repertoire significantly divergent from that of Pseudomonas syringae pv. tomato DC3000.

Diverse gene products including phytotoxins, pathogen-associated molecular patterns, and type III secreted effectors influence interactions between Pseudomonas syringae strains and plants, with additional yet uncharacterized factors likely contributing as well. Of particular interest are those interactions governing pathogen-host specificity. Comparative genomics of closely related pathogens with different host specificity represents an excellent approach for identification of genes contributing to host-range determination. A draft genome sequence of Pseudomonas syringae pv. tomato T1, which is pathogenic on tomato but nonpathogenic on Arabidopsis thaliana, was obtained for this purpose and compared with the genome of the closely related A. thaliana and tomato model pathogen P. syringae pv. tomato DC3000. Although the overall genetic content of each of the two genomes appears to be highly similar, the repertoire of effectors was found to diverge significantly. Several P. syringae pv. tomato T1 effectors absent from strain DC3000 were confirmed to be translocated into plants, with the well-studied effector AvrRpt2 representing a likely candidate for host-range determination. However, the presence of avrRpt2 was not found sufficient to explain A. thaliana resistance to P. syringae pv. tomato T1, suggesting that other effectors and possibly type III secretion system-independent factors also play a role in this interaction.

Mining the genomes of plant pathogenic bacteria: how not to drown in gigabases of sequence.

Hundreds of bacterial genomes including the genomes of dozens of plant pathogenic bacteria have been sequenced. These genomes represent an invaluable resource for molecular plant pathologists. In this review, we describe different approaches that can be used for mining bacterial genome sequences and examples of how some of these approaches have been used to analyse plant pathogen genomes so far. We review how genomes can be mined one by one and how comparative genomics of closely related genomes releases the true power of genomics. Databases and tools useful for genome mining that are publicly accessible on the Internet are also described. Finally, the need for new databases and tools to efficiently mine today's plant pathogen genomes and hundreds more in the near future is discussed.

Role of recombination in the evolution of the model plant pathogen Pseudomonas syringae pv. tomato DC3000, a very atypical tomato strain.

Pseudomonas syringae pv. tomato strain DC3000 (PtoDC3000) is one of the most intensively studied bacterial plant pathogens today. Here we report a thorough investigation into PtoDC3000 and close relatives isolated from Antirrhinum majus (snapdragon), Apium graveolens (celery), and Solanaceae and Brassicaceae species. Multilocus sequence typing (MLST) was used to resolve the precise phylogenetic relationship between isolates and to determine the importance of recombination in their evolution. MLST data were correlated with an analysis of the locus coding for the type III secreted (T3S) effector AvrPto1 to investigate the role of recombination in the evolution of effector repertoires. Host range tests were performed to determine if closely related isolates from different plants have different host ranges. It was found that PtoDC3000 is located in the same phylogenetic cluster as isolates from several Brassicaceae and Solanaceae species and that these isolates have a relatively wide host range that includes tomato, Arabidopsis thaliana, and cauliflower. All other analyzed tomato isolates from three different continents form a distinct cluster and are pathogenic only on tomato. Therefore, PtoDC3000 is a very unusual tomato isolate. Several recombination breakpoints were detected within sequenced gene fragments, and population genetic tests indicate that recombination contributed more than mutation to the variation between isolates. Moreover, recombination may play an important role in the reassortment of T3S effectors between strains. The data are finally discussed from a taxonomic standpoint, and P. syringae pv. tomato is proposed to be divided into two pathovars.

Naturally occurring nonpathogenic isolates of the plant pathogen Pseudomonas syringae lack a type III secretion system and effector gene orthologues.

Pseudomonas syringae causes plant diseases, and the main virulence mechanism is a type III secretion system (T3SS) that translocates dozens of effector proteins into plant cells. Here we report the existence of a subgroup of P. syringae isolates that do not cause disease on any plant species tested. This group is monophyletic and most likely evolved from a pathogenic P. syringae ancestor through loss of the T3SS. In the nonpathogenic isolate P. syringae 508 the genomic region that in pathogenic P. syringae strains contains the hrp-hrc cluster coding for the T3SS and flanking effector genes is absent. P. syringae 508 was also surveyed for the presence of effector orthologues from the closely related pathogenic strain P. syringae pv. syringae B728a, but none were detected. The absence of the hrp-hrc cluster and effector orthologues was confirmed for other nonpathogenic isolates. Using the AvrRpt2 effector as reporter revealed the inability of P. syringae 508 to translocate effectors into plant cells. Adding a plasmid-encoded T3SS and the P. syringae pv. syringae 61 effector gene hopA1 increased in planta growth almost 10-fold. This suggests that P. syringae 508 supplemented with a T3SS could be used to determine functions of individual effectors in the context of a plant infection, avoiding the confounding effect of other effectors with similar functions present in effector mutants of pathogenic isolates.

The life history of the plant pathogen Pseudomonas syringae is linked to the water cycle.

Pseudomonas syringae is a plant pathogen well known for its capacity to grow epiphytically on diverse plants and for its ice-nucleation activity. The ensemble of its known biology and ecology led us to postulate that this bacterium is also present in non-agricultural habitats, particularly those associated with water. Here, we report the abundance of P. syringae in rain, snow, alpine streams and lakes and in wild plants, in addition to the previously reported abundance in epilithic biofilms. Each of these substrates harbored strains that corresponded to P. syringae in terms of biochemical traits, pathogenicity and pathogenicity-related factors and that were ice-nucleation active. Phylogenetic comparisons of sequences of four housekeeping genes of the non-agricultural strains with strains of P. syringae from disease epidemics confirmed their identity as P. syringae. Moreover, strains belonging to the same clonal lineage were isolated from snow, irrigation water and a diseased crop plant. Our data suggest that the different substrates harboring P. syringae modify the structure of the associated populations. Here, we propose a comprehensive life cycle for P. syringae--in agricultural and non-agricultural habitats--driven by the environmental cycle of water. This cycle opens the opportunity to evaluate the importance of non-agricultural habitats in the evolution of a plant pathogen and the emergence of virulence. The ice-nucleation activity of all strains from snow, unlike from other substrates, strongly suggests that P. syringae plays an active role in the water cycle as an ice nucleus in clouds.