Leucine rich repeat-malectin receptor kinases IGP1/CORK1, IGP3 and IGP4 are required for arabidopsis immune responses triggered by ß-1,4-D-Xylo-oligosaccharides from plant cell walls.

Pattern-Triggered Immunity (PTI) in plants is activated upon recognition by Pattern Recognition Receptors (PRRs) of Damage- and Microbe-Associated Molecular Patterns (DAMPs and MAMPs) from plants or microorganisms, respectively. An increasing number of identified DAMPs/MAMPs are carbohydrates from plant cell walls and microbial extracellular layers, which are perceived by plant PRRs, such as LysM and Leucine Rich Repeat-Malectin (LRR-MAL) receptor kinases (RKs). LysM-RKs (e.g. CERK1, LYK4 and LYK5) are needed for recognition of fungal MAMP chitohexaose (ß-1,4-D-(GlcNAc)6, CHI6), whereas IGP1/CORK1, IGP3 and IGP4 LRR-MAL RKs are required for perception of ß-glucans, like cellotriose (ß-1,4-D-(Glc)3, CEL3) and mixed-linked glucans. We have explored the diversity of carbohydrates perceived by Arabidopsis thaliana seedlings by determining PTI responses upon treatment with different oligosaccharides and polysaccharides. These analyses revealed that plant oligosaccharides from xylans [ß-1,4-D-(xylose)4 (XYL4)], glucuronoxylans and α-1,4-glucans, and polysaccharides from plants and seaweeds activate PTI. Cross-elicitation experiments of XYL4 with other glycans showed that the mechanism of recognition of XYL4 and the DAMP 33-α-L-arabinofuranosyl-xylotetraose (XA3XX) shares some features with that of CEL3 but differs from that of CHI6. Notably, XYL4 and XA3XX perception is impaired in igp1/cork1, igp3 and igp4 mutants, and almost not affected in cerk1 lyk4 lyk5 triple mutant. XYL4 perception is conserved in different plant species since XYL4 pre-treatment triggers enhanced disease resistance in tomato to Pseudomonas syringae pv tomato DC3000 and PTI responses in wheat. These results expand the number of glycans triggering plant immunity and support IGP1/CORK1, IGP3 and IGP4 relevance in Arabidopsis thaliana glycans perception and PTI activation. Significance Statement: The characterization of plant immune mechanisms involved in the perception of carbohydrate-based structures recognized as DAMPs/MAMPs is needed to further understand plant disease resistance modulation. We show here that IGP1/CORK1, IGP3 and IGP4 LRR-MAL RKs are required for the perception of carbohydrate-based DAMPs ß-1,4-D-(xylose)4 (XYL4) and 33-α-L-arabinofuranosyl-xylotetraose (XA3XX), further expanding the function of these LRR-MAL RKs in plant glycan perception and immune activation.

Comparison of gene clustering criteria reveals intrinsic uncertainty in pangenome analyses.

BACKGROUND: A key step for comparative genomics is to group open reading frames into functionally and evolutionarily meaningful gene clusters. Gene clustering is complicated by intraspecific duplications and horizontal gene transfers that are frequent in prokaryotes. In consequence, gene clustering methods must deal with a trade-off between identifying vertically transmitted representatives of multicopy gene families, which are recognizable by synteny conservation, and retrieving complete sets of species-level orthologs. We studied the implications of adopting homology, orthology, or synteny conservation as formal criteria for gene clustering by performing comparative analyses of 125 prokaryotic pangenomes. RESULTS: Clustering criteria affect pangenome functional characterization, core genome inference, and reconstruction of ancestral gene content to different extents. Species-wise estimates of pangenome and core genome sizes change by the same factor when using different clustering criteria, allowing robust cross-species comparisons regardless of the clustering criterion. However, cross-species comparisons of genome plasticity and functional profiles are substantially affected by inconsistencies among clustering criteria. Such inconsistencies are driven not only by mobile genetic elements, but also by genes involved in defense, secondary metabolism, and other accessory functions. In some pangenome features, the variability attributed to methodological inconsistencies can even exceed the effect sizes of ecological and phylogenetic variables. CONCLUSIONS: Choosing an appropriate criterion for gene clustering is critical to conduct unbiased pangenome analyses. We provide practical guidelines to choose the right method depending on the research goals and the quality of genome assemblies, and a benchmarking dataset to assess the robustness and reproducibility of future comparative studies.

First confirmation of importation and transmission in Spain of the newly identified SARS-CoV-2 B.1.1.7 variant.

INTRODUCTION: A newly identified SARS-CoV-2 variant, VOC202012/01 originating lineage B.1.1.7, recently emerged in the United Kingdom. The rapid spread in the UK of this new variant has caused other countries to be vigilant. MATERIAL AND METHODS: We based our initial screening of B.1.1.7 on the dropout of the S gene signal in the TaqPath assay, caused by the 69/70 deletion. Subsequently, we confirmed the B.1.1.7 candidates by whole genome sequencing. RESULTS: We describe the first three imported cases of this variant from London to Madrid, subsequent post-arrival household transmission to three relatives, and the two first cases without epidemiological links to UK. One case required hospitalization. In all cases, drop-out of gene S was correctly associated to the B.1.1.7 variant, as all the corresponding sequences carried the 17 lineage-marker mutations. CONCLUSION: The first identifications of the SARS-CoV-2 B.1.1.7 variant in Spain indicate the role of independent introductions from the UK coexisting with post-arrival transmission in the community, since the early steps of this new variant in our country.

Genomic Analysis of Ceftazidime/Avibactam-Resistant GES-Producing Sequence Type 235 Pseudomonas aeruginosa Isolates.

The emergence of ceftazidime/avibactam (CZA) resistance among Guiana extended-spectrum ß-lactamase (GES)-producing Pseudomonas aeruginosa isolates has rarely been described. Herein, we analyze the phenotypic and genomic characterization of CZA resistance in different GES-producing P. aeruginosa isolates that emerged in our institution. A subset of nine CZA-resistant P. aeruginosa isolates was analyzed and compared with thirteen CZA-susceptible isolates by whole-genome sequencing (WGS). All CZA-resistant isolates belonged to the ST235 clone and O11 serotype. A variety of GES enzymes were detected: GES-20 (55.6%, 5/9), GES-5 (22.2%, 2/9), GES-1 (11.1%, 1/9), and GES-7 (11.1%, 1/9). WGS revealed the presence of two mutations within the blaGES-20 gene comprising two single-nucleotide substitutions, which caused aspartic acid/serine and leucine/premature stop codon amino acid changes at positions 165 (D165S) and 237 (L237X), respectively. No major differences in the mutational resistome (AmpC, OprD porin, and MexAB-OprM efflux pump-encoding genes) were found among CZA-resistant and CZA-susceptible isolates. None of the mutations that have been previously demonstrated to cause CZA resistance were observed. Different mutations within the blaGES-20 gene were documented in CZA-resistant GES-producing P. aeruginosa isolates belonging to the ST235 clone in our institution. Although further analysis should be performed, according to our results, other resistance mechanisms might be involved in CZA resistance.

Clinical and bacterial characteristics of Pseudomonas aeruginosa affecting the outcome of patients with bacteraemic pneumonia.

Few studies have assessed the clinical and bacterial characteristics of Pseudomonas aeruginosa (PA) bacteraemic pneumonia (BP) episodes. This study analysed all non-duplicate PA-BP episodes from a tertiary hospital in 2013-2017. Epidemiology, clinical data, antimicrobial therapy and outcomes were recorded. Whole-genome sequencing was performed on PA blood isolates. The impact on early and late overall mortality of host, antimicrobial treatment and pathogen factors was assessed by multivariate logistic regression analysis. Of 55 PA-BP episodes, 32 (58.2%) were caused by extensively drug-resistant (XDR) PA. ST175 (32.7%) and ST235 (25.5%) were the most frequent high-risk clones. ß-Lactamases/carbapenemases were detected in 29 isolates, including blaVIM-2 (27.2%) and blaGES type (25.5%) [blaGES-5 (20.0%), blaGES-1 (3.6%) and blaGES-20 (1.8%)]. The most prevalent O-antigen serotypes were O4 (34.5%) and O11 (30.9%). Overall, an extensive virulome was identified in all isolates. Early mortality (56.4%) was independently associated with severe neutropenia (aOR = 4.64, 95% CI 1.11-19.33; P = 0.035) and inappropriate empirical antimicrobial therapy (aOR = 5.71, 95% CI 1.41-22.98; P = 0.014). Additionally, late mortality (67.3%) was influenced by septic shock (aOR = 8.85, 95% CI 2.00-39.16; P = 0.004) and XDR phenotype (aOR = 5.46, 95% CI 1.25-23.85; P = 0.024). Moreover, specific genetic backgrounds [ST235, blaGES, gyrA (T83I), parC (S87L), exoU and O11 serotype] showed significant differences in patient outcomes. Our results confirm the high mortality associated with PA-BP. Besides relevant clinical characteristics and inappropriate empirical therapy, bacteria-specific genetics factors, such as XDR phenotype, adversely affect the outcome of PA-BP.

Genomic Epidemiology of SARS-CoV-2 in Madrid, Spain, during the First Wave of the Pandemic: Fast Spread and Early Dominance by D614G Variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in Madrid, Spain, on 25 February 2020. It increased in frequency very fast and by the end of May more than 70,000 cases had been confirmed by reverse transcription-polymerase chain reaction (RT-PCR). To study the lineages and the diversity of the viral population during this first epidemic wave in Madrid we sequenced 224 SARS-CoV-2 viral genomes collected from three hospitals from February to May 2020. All the known major lineages were found in this set of samples, though B.1 and B.1.5 were the most frequent ones, accounting for more than 60% of the sequences. In parallel with the B lineages and sublineages, the D614G mutation in the Spike protein sequence was detected soon after the detection of the first coronavirus disease 19 (COVID-19) case in Madrid and in two weeks became dominant, being found in 80% of the samples and remaining at this level during all the study periods. The lineage composition of the viral population found in Madrid was more similar to the European population than to the publicly available Spanish data, underlining the role of Madrid as a national and international transport hub. In agreement with this, phylodynamic analysis suggested multiple independent entries before the national lockdown and air transportation restrictions.

First confirmation of importation and transmission in Spain of the newly identified SARS-CoV-2 B.1.1.7 variant.

INTRODUCTION: A newly identified SARS-CoV-2 variant, VOC202012/01 originating lineage B.1.1.7, recently emerged in the United Kingdom. The rapid spread in the UK of this new variant has caused other countries to be vigilant. MATERIAL AND METHODS: We based our initial screening of B.1.1.7 on the dropout of the S gene signal in the TaqPath assay, caused by the 69/70 deletion. Subsequently, we confirmed the B.1.1.7 candidates by whole genome sequencing. RESULTS: We describe the first three imported cases of this variant from London to Madrid, subsequent post-arrival household transmission to three relatives, and the two first cases without epidemiological links to UK. One case required hospitalization. In all cases, drop-out of gene S was correctly associated to the B.1.1.7 variant, as all the corresponding sequences carried the 17 lineage-marker mutations. CONCLUSION: The first identifications of the SARS-CoV-2 B.1.1.7 variant in Spain indicate the role of independent introductions from the UK coexisting with post-arrival transmission in the community, since the early steps of this new variant in our country.

Free-living turtles are a reservoir for Salmonella but not for Campylobacter.

Different studies have reported the prevalence of Salmonella in turtles and its role in reptile-associated salmonellosis in humans, but there is a lack of scientific literature related with the epidemiology of Campylobacter in turtles. The aim of this study was to evaluate the prevalence of Campylobacter and Salmonella in free-living native (Emys orbicularis, n=83) and exotic (Trachemysscripta elegans, n=117) turtles from 11 natural ponds in Eastern Spain. In addition, different types of samples (cloacal swabs, intestinal content and water from Turtle containers) were compared. Regardless of the turtle species, natural ponds where individuals were captured and the type of sample taken, Campylobacter was not detected. Salmonella was isolated in similar proportions in native (8.0 ± 3.1%) and exotic (15.0 ± 3.3%) turtles (p=0.189). The prevalence of Salmonella positive turtles was associated with the natural ponds where animals were captured. Captured turtles from 8 of the 11 natural ponds were positive, ranged between 3.0 ± 3.1% and 60.0 ± 11.0%. Serotyping revealed 8 different serovars among four Salmonella enterica subspecies: S. enterica subsp. enterica (n = 21), S. enterica subsp. salamae (n = 2), S. enterica subsp. diarizonae (n = 3), and S. enterica subsp. houtenae (n = 1). Two serovars were predominant: S. Thompson (n=16) and S. typhimurium (n=3). In addition, there was an effect of sample type on Salmonella detection. The highest isolation of Salmonella was obtained from intestinal content samples (12.0 ± 3.0%), while lower percentages were found for water from the containers and cloacal swabs (8.0 ± 2.5% and 3.0 ± 1.5%, respectively). Our results imply that free-living turtles are a risk factor for Salmonella transmission, but do not seem to be a reservoir for Campylobacter. We therefore rule out turtles as a risk factor for human campylobacteriosis. Nevertheless, further studies should be undertaken in other countries to confirm these results.