Two Plant Bacteria, S. meliloti and Ca. Liberibacter asiaticus, Share Functional znuABC Homologues That Encode for a High Affinity Zinc Uptake System

The Znu system, encoded for by znuABC, can be found in multiple genera of bacteria and has been shown to be responsible for the import of zinc under low zinc conditions. Although this high-affinity uptake system is known to be important for both growth and/or pathogenesis in bacteria, it has not been functionally characterized in a plant-associated bacterium. A single homologue of this system has been identified in the plant endosymbiont, Sinorhizobium meliloti, while two homologous systems were found in the destructive citrus pathogen, Candidatus Liberibacter asiaticus. To understand the role of these protein homologues, a complementation assay was devised allowing the individual genes that comprise the system to be assayed independently for their ability to reinstate a partially-inactivated Znu system. Results from the assays have demonstrated that although all of the genes from S. meliloti were able to restore activity, only one of the two Ca. Liberibacter asiaticus encoded gene clusters contained genes that were able to functionally complement the system. Additional analysis of the gene clusters reveals that distinct modes of regulation may also exist between the Ca. Liberibacter asiaticus and S. meliloti import systems despite the intracellular-plant niche common to both of these bacteria

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Deciphering the Bacterial Microbiome of Citrus Plants in Response to ‘<i>Candidatus</i> Liberibacter asiaticus’-Infection and Antibiotic Treatments

<div><p>The bacterial microbiomes of citrus plants were characterized in response to ‘<i>Candidatus</i> Liberibacter asiaticus’ (Las)-infection and treatments with ampicillin (Amp) and gentamicin (Gm) by Phylochip-based metagenomics. The results revealed that 7,407 of over 50,000 known Operational Taxonomic Units (OTUs) in 53 phyla were detected in citrus leaf midribs using the PhyloChip™ G3 array, of which five phyla were dominant, <i>Proteobacteria</i> (38.7%), <i>Firmicutes</i> (29.0%), <i>Actinobacteria</i> (16.1%), <i>Bacteroidetes</i> (6.2%) and <i>Cyanobacteria</i> (2.3%). The OTU62806, representing ‘<i>Candidatus</i> Liberibacter’, was present with a high titer in the plants graft-inoculated with Las-infected scions treated with Gm at 100 mg/L and in the water-treated control (CK₁). However, the Las bacterium was not detected in the plants graft-inoculated with Las-infected scions treated with Amp at 1.0 g/L or in plants graft-inoculated with Las-free scions (CK₂). The PhyloChip array demonstrated that more OTUs, at a higher abundance, were detected in the Gm-treated plants than in the other treatment and the controls. Pairwise comparisons indicated that 23 OTUs from the <i>Achromobacter</i> spp. and 12 OTUs from the <i>Methylobacterium</i> spp. were more abundant in CK₂ and CK₁, respectively. Ten abundant OTUs from the <i>Stenotrophomonas</i> spp. were detected only in the Amp-treatment. These results provide new insights into microbial communities that may be associated with the progression of citrus huanglongbing (HLB) and the potential effects of antibiotics on the disease and microbial ecology.</p></div

Huanglongbing symptoms of the inoculated plants.

<p>Healthy grapefruit rootstocks graft-inoculated with HLB-affected lemon scions treated with ampicillin at a concentration of 1.0 g/L (<b>Amp</b>), gentamicin at a concentration of 100 mg/L (<b>Gm</b>), and a water control (disease control, <b>CK₁</b>); The healthy plants were graft-inoculated with Las-free lemon scions (healthy control, <b>CK₂</b>).</p

Distribution of the bacterial OTUs in response to antibiotic treatments.

<p>In the Venn diagram, the numbers in parentheses represent the number of bacterial OTUs that occurred in each antibiotic treatment [ampicillin (Amp) and gentamicin (Gm)], disease controls (CK₁), healthy control (CK₂) and their intersections. Pie charts A to G correspond to the appropriately labeled Venn diagram areas (A₁ to G₁ for the Amp treatment and A₂ to G₂ for the Gm treatment) and show families that contained over 1% of the total OTUs in each area. In pie charts A to G, the names of the families are followed by their frequencies as a percentage (%).</p

<i>Ca.</i> L. asiaticus (Las) and its transmission in grapefruit graft-inoculated with Las-infected lemon scions treated with ampicillin at 1.0 g/L (Amp), gentamicin at 100 mg/L (Gm), or water (disease control, CK₁) as well as grafted with the Las-free lemon scions (healthy control, CK₂).

§<p>Data were analyzed by a generalized linear mixed model using the SAS procedure GLIMMIX. Differences among treatment levels were determined with the LINES option of the LSMEANS statement. Different letter showed the significant difference at 0.05 levels (<i>Pr</i>≤0.05).</p

Bacterial community of leaf midribs of scions.

<p><b>A</b>) Composition and <b>B</b>) relative abundance of the bacterial Operational Taxonomic Units (OTUs) present in leaf midribs of scions from grapefruit rootstocks grafted with HLB-affected lemon scions treated with ampicillin (Amp), gentamicin (Gm) and water (disease control; CK₁). The healthy plants were grafted using Las-free lemon scions (healthy control; CK₂).</p

Sequence alignment of ZnuA orthologues. Orthologues to ZnuA from <i>S. meliloti</i> and <i>Ca.</i> Liberibacter asiaticus were aligned to ZnuA from <i>E. coli</i> using ClustalW.

<p>Residues that are 100% conserved amongst all orthologues are shown in bold. The three conserved His residues involved in binding zinc are shaded gray.</p

Bioinformatic analysis of the components comprising the Znu gene clusters.

a<p>In accordance with the database at the National Center for Biotechnology Information.</p>b<p>Percentages are based upon a ClustalW alignment between the corresponding protein in <i>E. coli.</i></p

Growth of <i>S. meliloti</i> complemented strains.

<p>Deletion mutants of <i>S. meliloti</i> for <b>A)</b><i>znuA</i> (ΔRZA), <b>B)</b><i>znuB</i> (ΔRZB), or <b>C)</b><i>znuC</i> (ΔRZC) were complemented with the corresponding <i>znu</i> genes from either the wild-type <i>S. meliloti</i> (orange bars), or <i>Ca.</i> Liberibacter asiaticus (blue bars). As controls, the wild-type <i>S. meliloti</i> strain (black bars), the uncomplemented deletion strains (white bars), and those containing the vector control (grey bars) were also grown. Strains were grown in different concentrations of EDTA ranging from 0 to 0.2 mM (x-axis). The absorbance at 600 nm (OD₆₀₀) was measured at the time points indicated. Data shown is an average of three independent replicates with error bars representing the standard deviation.</p