Genetically divergent Francisella philomiragia associated with septic arthritis, Montevideo, Uruguay.

Here we report a case of septic arthritis associated with a genetically divergent Francisella philomiragia strain in a patient with chronic rheumatoid arthritis and Adult-onset Still's disease (AOSD) in Maldonado, Uruguay. In this study mass spectrometry together with whole-genome sequencing using Oxford Nanopore technology allowed for the correct identification of the etiologic agent.

Erratum: Citrus genomic resources unravel putative genetic determinants of Huanglongbing pathogenicity.

[This corrects the article DOI: 10.1016/j.isci.2023.106024.].

An antibiotic produced by Pseudomonas fluorescens CFBP2392 with antifungal activity against Rhizoctonia solani.

Pseudomonas fluorescens CFBP2392 has been recognized as a potential biocontrol agent due to its ability to suppress damping-off and root rot disease. This isolate has antibacterial activity in vitro as many other strains from the Pseudomonas fluorescens complex. In this work, the antibacterial and antifungal activity of the strain were explored. Dual culture assays evidenced the antifungal activity of the strain against different phytopathogens: Alternaria sp., Pythium ultimun, Fusarium oxysporum, and Rhizoctonia solani. Purification of an antifungal fraction was performed by preparative HPLC from the chemical extraction of growth media. The fraction showed altered R. solani growth and ultrastructure. Transmission electron microscopy revealed the purified compound induced hypertrophied mitochondria, membranous vesicles, and a higher number of vacuoles in R. salani cytoplasm. In addition, co-cultivation of P. fluorescens CFBP2392 with R. solani resulted in an enlarged and deformed cell wall. To gain genomic insights on this inhibition, the complete genome of P. fluorescens CFBP2392 was obtained with Oxford Nanopore technology. Different biosynthetic gene clusters (BGCs) involved in specialized metabolites production including a lokisin-like and a koreenceine-like cluster were identified. In accordance with the putative BGCs identified, sequence phylogeny analysis of the MacB transporter in the lokisin-like cluster further supports the similarity with other transporters from the amphisin family. Our results give insights into the cellular effects of the purified microbial metabolite in R. solani ultrastructure and provide a genomic background to further explore the specialized metabolite potential.

The mechanism of the phage-encoded protein antibiotic from ΦX174.

The historically important phage ΦX174 kills its host bacteria by encoding a 91-residue protein antibiotic called protein E. Using single-particle electron cryo-microscopy, we demonstrate that protein E bridges two bacterial proteins to form the transmembrane YES complex [MraY, protein E, sensitivity to lysis D (SlyD)]. Protein E inhibits peptidoglycan biosynthesis by obstructing the MraY active site leading to loss of lipid I production. We experimentally validate this result for two different viral species, providing a clear model for bacterial lysis and unifying previous experimental data. Additionally, we characterize the Escherichia coli MraY structure-revealing features of this essential enzyme-and the structure of the chaperone SlyD bound to a protein. Our structures provide insights into the mechanism of phage-mediated lysis and for structure-based design of phage therapeutics.

Citrus genomic resources unravel putative genetic determinants of Huanglongbing pathogenicity.

Citrus HLB caused by Candidatus Liberibacter asiaticus is a pathogen-triggered immune disease. Here, we identified putative genetic determinants of HLB pathogenicity by integrating citrus genomic resources to characterize the pan-genome of accessions that differ in their response to HLB. Genome-wide association mapping and analysis of allele-specific expression between susceptible, tolerant, and resistant accessions further refined candidates underlying the response to HLB. We first developed a phased diploid assembly of Citrus sinensis 'Newhall' genome and produced resequencing data for 91 citrus accessions that differ in their response to HLB. These data were combined with previous resequencing data from 356 accessions for genome-wide association mapping of the HLB response. Genes determinants for HLB pathogenicity were associated with host immune response, ROS production, and antioxidants. Overall, this study has provided a significant resource of citrus genomic data and identified candidate genes to be further explored to understand the genetic determinants of HLB pathogenicity.

Human microbiota drives hospital-associated antimicrobial resistance dissemination in the urban environment and mirrors patient case rates.

BACKGROUND: The microbial community composition of urban environments is primarily determined by human activity. The use of metagenomics to explore how microbial communities are shaped in a city provides a novel input that can improve decisions on public health measures, architectural design, and urban resilience. Of note, the sewage system in a city acts as a complex reservoir of bacteria, pharmaceuticals, and antimicrobial resistant (AMR) genes that can be an important source of epidemiological information. Hospital effluents are rich in patient-derived bacteria and can thus readily become a birthplace and hotspot reservoir for antibiotic resistant pathogens which are eventually incorporated into the environment. Yet, the scope to which nosocomial outbreaks impact the urban environment is still poorly understood. RESULTS: In this work, we extensively show that different urban waters from creeks, beaches, sewage spillways and collector pipes enclose discrete microbial communities that are characterized by a differential degree of contamination and admixture with human-derived bacteria. The abundance of human bacteria correlates with the abundance of AMR genes in the environment, with beta-lactamases being the top-contributing class to distinguish low vs. highly-impacted urban environments. Indeed, the abundance of beta-lactamase resistance and carbapenem resistance determinants in the urban environment significantly increased in a 1-year period. This was in line with a pronounced increase of nosocomial carbapenem-resistant infections reported during the same period that was mainly driven by an outbreak-causing, carbapenemase-producing Klebsiella pneumoniae (KPC) ST-11 strain. Genome-resolved metagenomics of urban waters before and after this outbreak, coupled with high-resolution whole-genome sequencing, confirmed the dissemination of the ST-11 strain and a novel KPC megaplasmid from the hospital to the urban environment. City-wide analysis showed that geospatial dissemination of the KPC megaplasmid in the urban environment inversely depended on the sewage system infrastructure. CONCLUSIONS: We show how urban metagenomics and outbreak genomic surveillance can be coupled to generate relevant information for infection control, antibiotic stewardship, and pathogen epidemiology. Our results highlight the need to better characterize and understand how human-derived bacteria and antimicrobial resistance disseminate in the urban environment to incorporate this information in the development of effluent treatment infrastructure and public health policies. Video Abstract.

The structure and function of the global citrus rhizosphere microbiome.

Citrus is a globally important, perennial fruit crop whose rhizosphere microbiome is thought to play an important role in promoting citrus growth and health. Here, we report a comprehensive analysis of the structural and functional composition of the citrus rhizosphere microbiome. We use both amplicon and deep shotgun metagenomic sequencing of bulk soil and rhizosphere samples collected across distinct biogeographical regions from six continents. Predominant taxa include Proteobacteria, Actinobacteria, Acidobacteria and Bacteroidetes. The core citrus rhizosphere microbiome comprises Pseudomonas, Agrobacterium, Cupriavidus, Bradyrhizobium, Rhizobium, Mesorhizobium, Burkholderia, Cellvibrio, Sphingomonas, Variovorax and Paraburkholderia, some of which are potential plant beneficial microbes. We also identify over-represented microbial functional traits mediating plant-microbe and microbe-microbe interactions, nutrition acquisition and plant growth promotion in citrus rhizosphere. The results provide valuable information to guide microbial isolation and culturing and, potentially, to harness the power of the microbiome to improve plant production and health.

Induced Systemic Resistance Against Citrus Canker Disease by Rhizobacteria.

Citrus canker, caused by Xanthomonas citri subsp. citri, is an important citrus disease that causes significant economic losses worldwide. All commercial citrus varieties are susceptible to citrus canker. Currently, chemical control with copper based products is the main approach to control X. citri subsp. citri dispersal and plant colonization. However, extensive use of copper compounds can result in copper-resistant strains and cause adverse effects on the environment. Alternatives to chemical control involve the activation of citrus immunity to control the disease. Here, we investigated the ability of multiple rhizobacteria to induce a systemic defense response in cultivar Duncan grapefruit. Burkholderia territorii strain A63, Burkholderia metallica strain A53, and Pseudomonas geniculata strain 95 were found to effectively activate plant defense and significantly reduce symptom development in leaves challenged with X. citri subsp. citri. In the priming phase, root application of P. geniculata induced the expression of salicylic acid (SA)-signaling pathway marker genes (PR1, PR2, PR5, and salicylic acid carboxyl methyltransferase [SAM-SACM]). Gene expression analyses after X. citri subsp. citri challenge showed that root inoculation with P. geniculata strain 95 increased the relative levels of phenylalanine ammonia lyase 1 and SAM-SACM, two genes involved in the phenylpropanoid pathway as well as the biosynthesis of SA and methyl salicylate (MeSA), respectively. However, hormone analyses by UPLC-MS/MS showed no significant difference between SA in P. geniculata-treated plants and control plants at 8 days post-beneficial bacteria root inoculation. Moreover, P. geniculata root-treated plants contained higher reactive oxygen species levels in aerial tissues than control plants 8 days post-treatment application. This study demonstrates that rhizobacteria can modulate citrus immunity resulting in a systemic defense response against X. citri subsp. citri under greenhouse conditions.

Huanglongbing impairs the rhizosphere-to-rhizoplane enrichment process of the citrus root-associated microbiome.

BACKGROUND: Roots are the primary site for plant-microbe interactions. Among the three root-associated layers (i.e., rhizosphere, rhizoplane, and endorhiza), the rhizoplane is a key component serving a critical gating role that controls microbial entry into plant roots. The microbial communities colonizing the three layers are believed to be gradually enriched from the bulk soil inoculum. However, it is unknown how this enrichment process, particularly the rhizosphere to rhizoplane step, is affected by biotic stresses, such as disease. In this study, we address this question using the citrus root-associated microbiome as a model. RESULTS: We identified the rhizosphere-to-rhizoplane-enriched taxonomic and functional properties of the citrus root-associated microbiome and determined how they were affected by Huanglongbing (HLB), a severe systemic disease caused by Candidatus Liberibacter asiaticus, using metagenomic and metatranscriptomic approaches. Multiple rhizoplane-enriched genera were identified, with Bradyrhizobium and Burkholderia being the most dominant. Plant-derived carbon sources are an important driving force for the enrichment process. The enrichment of functional attributes, such as motility, chemotaxis, secretion systems, and lipopolysaccharide (LPS) synthesis, demonstrated more active microbe-plant interactions on the rhizoplane than the rhizosphere. We observed that HLB impaired the rhizosphere-to-rhizoplane enrichment process of the citrus root-associated microbiome in three ways: (1) by decreasing the relative abundance of most rhizoplane-enriched genera; (2) by reducing the relative abundance and/or expression activity of the functional attributes involved in microbe-plant interactions; and (3) by recruiting more functional features involved in autotrophic life cycle adaptation, such as carbon fixation and nitrogen nitrification in the HLB rhizoplane microbiome. Finally, our data showed that inoculation of Burkholderia strains isolated from the healthy citrus root-associated microbiome could trigger the expression of genes involved in induced systemic resistance in inoculated plants. CONCLUSIONS: HLB causes decreased relative abundance and/or expression activity of rhizoplane-enriched taxonomic and functional properties, collectively resulting in impaired plant host-microbiome interactions. Manipulation of the citrus root-associated microbiome, for instance, by inoculating citrus roots with beneficial Burkholderia strains, has potential to promote plant health. Our results provide novel insights for understanding the contributions of the community enrichment process of the root-associated microbiome to the plant hosts.

Characterization of Antimicrobial-Producing Beneficial Bacteria Isolated from Huanglongbing Escape Citrus Trees.

The microbiome associated with crop plants has a strong impact on their health and productivity. Candidatus Liberibacter asiaticus (Las), the bacterial pathogen responsible for Huanglongbing (HLB) disease, lives inside the phloem of citrus plants including the root system. It has been suggested that Las negatively affects citrus microbiome. On the other hand, members of citrus microbiome also influence the interaction between Las and citrus. Here, we report the isolation and characterization of multiple putative beneficial bacteria from healthy citrus rhizosphere. Firstly, six bacterial strains showing antibacterial activity against two bacteria closely related to Las: Agrobacterium tumefaciens and Sinorhizobium meliloti were selected. Among them, Burkholderia metallica strain A53 and Burkholderia territorii strain A63 are within the ß-proteobacteria class, whereas Pseudomonas granadensis strain 100 and Pseudomonas geniculata strain 95 are within the γ-proteobacteria class. Additionally, two gram-positive bacteria Rhodococcus jialingiae strain 108 and Bacillus pumilus strain 104 were also identified. Secondly, antimicrobial activity against three fungal pathogens: Alternaria alternata, Colletotrichum acutatum, Phyllosticta citricarpa, and two oomycetes: Phytophthora nicotianae and Phytophthora palmivora. Four bacterial strains Burkholderia territorii A63, Burkholderia metallica A53, Pseudomonas geniculata 95, and Bacillus pumilus 104 were shown to have antagonistic activity against the citrus root pathogen Phytophthora nicotianae based on dual culture antagonist assays and compartmentalized petri dish assays. The four selected bacteria were sequenced. Genes involved in phosphate solubilization, siderophore production and iron acquisition, volatile organic compound production, osmoprotection and osmotic tolerance, phytohormone production, antagonism, and nutrient competition were predicted and discussed related to the beneficial traits.

Repertoire of novel sequence signatures for the detection of Candidatus Liberibacter asiaticus by quantitative real-time PCR.

BACKGROUND: Huanglongbing (HLB) or citrus greening is a devastating disease of citrus. The gram-negative bacterium Candidatus Liberibacter asiaticus (Las) belonging to the α-proteobacteria is responsible for HLB in North America as well as in Asia. Currently, there is no cure for this disease. Early detection and quarantine of Las-infected trees are important management strategies used to prevent HLB from invading HLB-free citrus producing regions. Quantitative real-time PCR (qRT-PCR) based molecular diagnostic assays have been routinely used in the detection and diagnosis of Las. The oligonucleotide primer pairs based on conserved genes or regions, which include 16S rDNA and the ß-operon, have been widely employed in the detection of Las by qRT-PCR. The availability of whole genome sequence of Las now allows the design of primers beyond the conserved regions for the detection of Las explicitly. RESULTS: We took a complimentary approach by systematically screening the genes in a genome-wide fashion, to identify the unique signatures that are only present in Las by an exhaustive sequence based similarity search against the nucleotide sequence database. Our search resulted in 34 probable unique signatures. Furthermore, by designing the primer pair specific to the identified signatures, we showed that most of our primer sets are able to detect Las from the infected plant and psyllid materials collected from the USA and China by qRT-PCR. Overall, 18 primer pairs of the 34 are found to be highly specific to Las with no cross reactivity to the closely related species Ca. L. americanus (Lam) and Ca. L. africanus (Laf). CONCLUSIONS: We have designed qRT-PCR primers based on Las specific genes. Among them, 18 are suitable for the detection of Las from Las-infected plant and psyllid samples. The repertoire of primers that we have developed and characterized in this study enhanced the qRT-PCR based molecular diagnosis of HLB.