Erwinia species identification using matrix-assisted laser desorption ionization-time of flight mass spectrometry

Rapid and reliable identification of plant pathogenic bacteria is critical for effective implementation of phytosanitary measures. The genus Erwinia includes a number of economically important plant pathogens such as fire blight agent Erwinia amylovora or Asian pear pathogen Erwinia pyrifoliae, together with closely related plant epiphytes of unknown pathogenicity or even with a potential use for biological control like Erwinia tasmaniensis or Erwinia billingiae, respectively. Current laboratory methods to achieve satisfactory identification and discrimination between species within the Erwinia genus are based on the isolation on semi-selective media, serology, specific PCR and gene locus sequencing: these approaches are complicated and time-consuming, often requiring a priori assumptions over the identity of the isolates. Here we present a streamlined approach based on whole-cell Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS) based on the AXIMA mass spectrometer of Shimadzu-Biotech Corp that demonstrates the potential of this technology for quick species identification in plant diagnostics within the genus Erwinia

Surgical management of a life-threatening retro-pharyngeal haematoma following trans-oesophageal echocardiography = Trattamento chirurgico di un pericoloso ematoma retrofaringeo, sviluppatosi a seguito di un\u2019ecocardiografia transesofagea

Nonostante siano state descritte complicanze potenzialmente letali, l\u2019ecocardiografia transesofagea viene considerata una procedura diagnostica sicura. Scopo del presente lavoro \ue8 sottolineare come, nei casi di voluminosi ematomi secondari a tale procedura, possa diventare indispensabile il ricorso alla chirurgia per tutelare la vita del paziente. Nel caso qui descritto, un paziente affetto da spondilosi cervicale e in terapia anticoagulante ha sviluppato, a seguito di un\u2019ecocardiografia transesofagea, un ematoma retrofaringeo tanto voluminoso da determinare un\u2019ostruzione delle vie aeree superiori. Per assicurarne la perviet\ue0 \ue8 stato necessario ricorrere a una tracheotomia, l\u2019ematoma, invece, \ue8 stato drenato per via transcervicale. Il paziente \ue8 guarito completamente in 10 giorni. Il chirurgo otorinolaringoiatrico dovrebbe essere a conoscenza, non solo di questa rara complicanza, ma anche della necessit\ue0 di un suo trattamento chirurgico.Trans-oesophageal echocardiography is generally considered a safe procedure, but occasional life-threatening complications have been reported. The aim of this clinical investigation is to outline the need of surgical management in cases of large retro-pharyngeal haematoma following trans-oesophageal echocardiography. In the case reported here, a patient with cervical spondylosis on anti-coagulant therapy was referred to the Head and Neck Department because of a retro-pharyngeal haematoma with severe upper airway obstruction following transoesophageal echocardiography. Tracheotomy was required to guarantee respiratory function, while trans-cervical surgery was performed to evacuate the haematoma. Total recovery was achieved within 10 days. In conclusion, the head and neck surgeon should consider the need of surgical management in cases of retro-pharyngeal haematoma following trans-oesophageal echocardiography

Comparative genomic analysis of the biotechnological potential of the novel species Pseudomonas wadenswilerensis CCOS 864T and Pseudomonas reidholzensis CCOS 865T

In recent years, the use of whole-cell biocatalysts and biocatalytic enzymes in biotechnological applications originating from the genus Pseudomonas has greatly increased. In 2014, two new species within the Pseudomonas putida group were isolated from Swiss forest soil. In this study, the high quality draft genome sequences of Pseudomonas wadenswilerensis CCOS 864T and Pseudomonas reidholzensis CCOS 865T were used in a comparative genomics approach to identify genomic features that either di ered between these two new species or to selected members of the P. putida group. The genomes of P. wadenswilerensis CCOS 864T and P. reidholzensis CCOS 865T were found to share genomic features for the degradation of aromatic compounds or the synthesis of secondary metabolites. In particular, genes encoding for biocatalytic relevant enzymes belonging to the class of oxidoreductases, proteases and isomerases were found, that could yield potential applications in biotechnology. Ecologically relevant features revealed that both species are probably playing an important role in the degradation of soil organic material, the accumulation of phosphate and biocontrol against plant pathogens

Genome-based population structure analysis of the strawberry plant pathogen Xanthomonas fragariae reveals two distinct groups that evolved independently before its species description

Xanthomonas fragariae is a quarantine organism in Europe, causing angular leaf spots on strawberry plants. It is spreading worldwide in strawberry-producing regions due to import of plant material through trade and human activities. In order to resolve the population structure at the strain level, we have employed high-resolution molecular typing tools on a comprehensive strain collection representing global and temporal distribution of the pathogen. Clustered regularly interspaced short palindromic repeat regions (CRISPRs) and variable number of tandem repeats (VNTRs) were identified within the reference genome of X. fragariae LMG 25863 as a potential source of variation. Strains from our collection were whole-genome sequenced and used in order to identify variable spacers and repeats for discriminative purpose. CRISPR spacer analysis and multiple-locus VNTR analysis (MLVA) displayed a congruent population structure, in which two major groups and a total of four subgroups were revealed. The two main groups were genetically separated before the first X. fragariae isolate was described and are potentially responsible for the worldwide expansion of the bacterial disease. Three primer sets were designed for discriminating CRISPR-associated markers in order to streamline group determination of novel isolates. Overall, this study describes typing methods to discriminate strains and monitor the pathogen population structure, more especially in the view of a new outbreak of the pathogen

High-quality draft genome sequence of Pseudomonas reidholzensis strain CCOS 865T

We have sequenced and assembled the genome of Pseudomonas reidholzensis CCOS 865T, which was isolated in 2014 from forest soil. Members of the genus Pseudomonas play important roles in environmental systems and are utilized in many biotechnological processes. The genome of this species may provide an important resource for the discovery of novel enzyme activities

Synergistic interaction between the type III secretion system of the endophytic bacterium <i>Pantoea agglomerans</i> DAPP-PG 734 and the virulence of the causal agent of olive knot <i>Pseudomonas savastanoi pv. savastanoi</i> DAPP-PG 722

The endophytic bacterium Pantoea agglomerans DAPP-PG 734 was previously isolated from olive knots caused by infection with Pseudomonas savastanoi pv. savastanoi DAPP-PG 722. Whole-genome analysis of this P. agglomerans strain revealed the presence of a Hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS). To assess the role of the P. agglomerans T3SS in the interaction with P. savastanoi pv. savastanoi, we generated independent knockout mutants in three Hrp genes of the P. agglomerans DAPP-PG 734 T3SS (hrpJ, hrpN, and hrpY). In contrast to the wildtype control, all three mutants failed to cause a hypersensitive response when infiltrated in tobacco leaves, suggesting that P. agglomerans T3SS is functional and injects effector proteins in plant cells. In contrast to P. savastanoi pv. savastanoi DAPP-PG 722, the wildtype strain P. agglomerans DAPP-PG 734 and its Hrp T3SS mutants did not cause olive knot disease in 1-year-old olive plants. Coinoculation of P. savastanoi pv. savastanoi with P. agglomerans wildtype strains did not significantly change the knot size, while the DAPP-PG 734 hrpY mutant induced a significant decrease in knot size, which could be complemented by providing hrpY on a plasmid. By epifluorescence microscopy and confocal laser scanning microscopy, we found that the localization patterns in knots were nonoverlapping for P. savastanoi pv. savastanoi and P. agglomerans when coinoculated. Our results suggest that suppression of olive plant defences mediated by the Hrp T3SS of P. agglomerans DAPP-PG 734 positively impacts the virulence of P. savastanoi pv. savastanoi DAPP-PG 722

Development and test of 21 multiplex PCRs composed of SSRs spanning most of the apple genome

A series of 21 multiplex (MP) polymerase chain reactions containing simple sequence repeat (SSR) markers spanning most of the apple genome has been developed. Eighty-eight SSR markers, well distributed over all 17 linkage groups (LGs), have been selected. Eighty-four of them were included in 21 different MPs while four could not be included in any MPs. The 21 MPs were then used to genotype approximately 2,000 DNA samples from the European High-quality Disease-Resistant Apples for a Sustainable agriculture project. Two SSRs (CH01d03 and NZAL08) were discarded at an early stage as they did not produce stable amplifications in the MPs, while the scoring of the multilocus (ML) SSR Hi07d11 and CN44794 was too complex for large-scale genotyping. The testing of the remaining 80 SSRs over a large number of different genotypes allowed: (1) a better estimation of their level of polymorphism; as well as of (2) the size range of the alleles amplified; (3) the identification of additional unmapped loci of some ML SSRs; (4) the development of methods to assign alleles to the different loci of ML SSRs and (5) conditions at which an SSR previously described as ML would amplify alleles of a single locus to be determined. These data resulted in the selection of 75 SSRs out of the 80 that are well suited and recommended for large genotyping project

Comparative genomics to examine the endophytic potential of Pantoea agglomerans DAPP-PG 734

Pantoea agglomerans DAPP-PG 734 was isolated as endophyte from knots (tumors) caused by Pseudomonas savastanoi pv. savastanoi DAPP-PG 722 in olive trees. To understand the plant pathogen-endophyte interaction on a genomic level, the whole genome of P. agglomerans DAPP-PG 734 was sequenced and annotated. The complete genome had a total size of 5′396′424 bp, containing one circular chromosome and four large circular plasmids. The aim of this study was to identify genomic features that could play a potential role in the interaction between P. agglomerans DAPP-PG 734 and P. savastanoi pv. savastanoi DAPP-PG 722. For this purpose, a comparative genomic analysis between the genome of P. agglomerans DAPP-PG 734 and those of related Pantoea spp. was carried out. In P. agglomerans DAPP-PG 734, gene clusters for the synthesis of the Hrp-1 type III secretion system (T3SS), type VI secretion systems (T6SS) and autoinducer, which could play an important role in a plant-pathogenic community enhancing knot formation in olive trees, were identified. Additional gene clusters for the biosynthesis of two different antibiotics, namely dapdiamide E and antibiotic B025670, which were found in regions between integrative conjugative elements (ICE), were observed. The in-depth analysis of the whole genome suggested a characterization of the P. agglomerans DAPP-PG 734 isolate as endophytic bacterium with biocontrol activity rather than as a plant pathogen

Molecular investigation of isolates from a multistate polymicrobial outbreak associated with contaminated total parenteral nutrition in Brazil

Background: Between November 2013 and June 2014, 56 cases of bacteremia (15 deaths) associated with the use of Total Parenteral Nutrition (TPN) and/or calcium gluconate (CG) were reported in four Brazilian states. Methods: We analyzed 73 bacterial isolates from four states: 45 from blood, 25 from TPN and three from CG, originally identified as Acinetobacter baumannii, Rhizobium radiobacter, Pantoea sp. or Enterobacteriaceae using molecular methods. Results: The first two bacterial species were confirmed while the third group of species could not be identified using standard identification protocols. These isolates were subsequently identified by Multi-Locus Sequence Analysis as Phytobacter diazotrophicus, a species related to strains from similar outbreaks in the United States in the 1970’s. Within each species, TPN and blood isolates proved to be clonal, whereas the R. radiobacter isolates retrieved from CG were found to be unrelated. Conclusion: This is the first report of a three-species outbreak caused by TPN contaminated with A. baumannii, R. radiobacter and P. diazotrophicus. The concomitant presence of clonal A. baumannii and P. diazotrophicus isolates in several TPN and blood samples, as well as the case of one patient, where all three different species were isolated simultaneously, suggest that the outbreak may be ascribed to a discrete contamination of TPN. In addition, this study highlights the clinical relevance of P. diazotrophicus, which has been involved in outbreaks in the past, but was often misidentified as P. agglomerans