Bacterial persistence to antibiotics activated by tRNA mutations.

Objectives: Bacterial persistence is a significant cause of the intractability of chronic and relapsing infections. Despite its importance, many of the underlying mechanisms are still not well understood.

Methods: Antibiotic-tolerant mutants of Burkholderia thailandensis were isolated through exposure to lethal doses of AMP or MEM, followed by whole-genome sequencing to identify mutations.

Mutations Affecting Cellular Levels of Cobalamin (Vitamin B) Confer Tolerance to Bactericidal Antibiotics in .

The complex (Bcc) consists of opportunistic pathogens known to cause pneumonia in immunocompromised individuals, especially those with cystic fibrosis. Treating Bcc pneumonia is challenging due to the pathogens' high multidrug resistance. Therefore, inhalation therapy with tobramycin powder, which can achieve high antibiotic concentrations in the lungs, is a promising treatment option.

A Two-Component-System-Governed Regulon That Includes a β-Lactamase Gene is Responsive to Cell Envelope Disturbance.

β-Lactamase production facilitates bacterial survival in nature and affects many infection therapies. However, much of its regulation remains unexplored. We used a genetics-based approach to identify a two-component system (TCS) present in a strain of Burkholderia thailandensis essential for the regulated expression of a class A β-lactamase gene, , by sensing subtle envelope disturbance caused by β-lactams, polymyxin B, or other chemical agents.

Do an Altered Gut Microbiota and an Associated Leaky Gut Affect COVID-19 Severity?

Coronavirus disease 2019 (COVID-19), which has been declared a pandemic, has exhibited a wide range of severity worldwide. Although this global variation is largely affected by socio-medical situations in each country, there is also high individual-level variation attributable to elderliness and certain underlying medical conditions, including high blood pressure, diabetes, and obesity. As both elderliness and the aforementioned chronic conditions are often associated with an altered gut microbiota, resulting in disrupted gut barrier integrity, and gut symptoms have consistently been associated with more severe illness in COVID-19 patients, it is possible that dysfunction of the gut as a whole influences COVID-19 severity.

Non-catalytic-Region Mutations Conferring Transition of Class A β-Lactamases Into ESBLs.

Despite class A ESBLs carrying substitutions outside catalytic regions, such as Cys69Tyr or Asn136Asp, have emerged as new clinical threats, the molecular mechanisms underlying their acquired antibiotics-hydrolytic activity remains unclear. We discovered that this non-catalytic-region (NCR) mutations induce significant dislocation of β3-β4 strands, conformational changes in critical residues associated with ligand binding to the lid domain, dynamic fluctuation of Ω-loop and β3-β4 elements. Such structural changes increase catalytic regions' flexibility, enlarge active site, and thereby accommodate third-generation cephalosporin antibiotics, ceftazidime (CAZ).

Mutations in ArgS Arginine-tRNA Synthetase Confer Additional Antibiotic Tolerance Protection to Extended-Spectrum-β-Lactamase-Producing Burkholderia thailandensis.

Highly conserved PenI-type class A β-lactamase in pathogenic members of species can evolve to extended-spectrum β-lactamase (ESBL), which exhibits hydrolytic activity toward third-generation cephalosporins, while losing its activity toward the original penicillin substrates. We describe three single-amino-acid-substitution mutations in the ArgS arginine-tRNA synthetase that confer extra antibiotic tolerance protection to ESBL-producing This pathway can be exploited to evade antibiotic tolerance induction in developing therapeutic measures against species, targeting their essential aminoacyl-tRNA synthetases.

Antibiotic Scars Left on the Gut Microbiota from the Stringent Response.

Current research is primarily focused on compositional shifts and alterations in the metabolic status of the gut microbiota to elucidate the damage caused by antibiotics. However, the impact of the stringent response, which is governed by a global gene regulatory system conserved in most gut bacteria, should not be overlooked.

Mutations in MetG (methionyl-tRNA synthetase) and TrmD [tRNA (guanine-N1)-methyltransferase] conferring meropenem tolerance in Burkholderia thailandensis.

Objectives: Although meropenem is widely used to treat Burkholderia infections, the response of Burkholderia pathogens to this antibiotic is largely unexplored.

Methods: Burkholderia thailandensis, a model for Burkholderia spp., particularly Burkholderia mallei and Burkholderia pseudomallei, was challenged with a lethal level of meropenem and survivors were isolated.

High adaptability of the omega loop underlies the substrate-spectrum-extension evolution of a class A β-lactamase, PenL.

The omega loop in β-lactamases plays a pivotal role in substrate recognition and catalysis, and some mutations in this loop affect the adaptability of the enzymes to new antibiotics. Various mutations, including substitutions, deletions, and intragenic duplications resulting in tandem repeats (TRs), have been associated with β-lactamase substrate spectrum extension. TRs are unique among the mutations as they cause severe structural perturbations in the enzymes.

Faecalibacterium prausnitzii subspecies-level dysbiosis in the human gut microbiome underlying atopic dermatitis.

Background: Atopic dermatitis (AD) is a serious global epidemic associated with a modern lifestyle.

Objective: Although aberrant interactions between gut microbes and the intestinal immune system have been implicated in this skin disease, the nature of the microbiome dysfunction underlying the disease remains unclear.

Methods: The gut microbiome from 132 subjects, including 90 patients with AD, was analyzed by using 16S rRNA gene and metagenome sequence analyses.

Cell Wall Recycling-Linked Coregulation of AmpC and PenB β-Lactamases through ampD Mutations in Burkholderia cenocepacia.

In many Gram-negative pathogens, mutations in the key cell wall-recycling enzyme AmpD (N-acetyl-anhydromuramyl-L-alanine amidase) affect the activity of the regulator AmpR, which leads to the expression of AmpC β-lactamase, conferring resistance to expanded-spectrum cephalosporin antibiotics. Burkholderia cepacia complex (Bcc) species also have these Amp homologs; however, the regulatory circuitry and the nature of causal ampD mutations remain to be explored. A total of 92 ampD mutants were obtained, representing four types of mutations: single nucleotide substitution (causing an amino acid substitution or antitermination of the enzyme), duplication, deletion, and IS element insertion.

The tandem repeats enabling reversible switching between the two phases of β-lactamase substrate spectrum.

Expansion or shrinkage of existing tandem repeats (TRs) associated with various biological processes has been actively studied in both prokaryotic and eukaryotic genomes, while their origin and biological implications remain mostly unknown. Here we describe various duplications (de novo TRs) that occurred in the coding region of a β-lactamase gene, where a conserved structure called the omega loop is encoded. These duplications that occurred under selection using ceftazidime conferred substrate spectrum extension to include the antibiotic.

Deletion mutations conferring substrate spectrum extension in the class A β-lactamase.

We describe four new deletion mutations in a class A β-lactamase PenA in Burkholderia thailandensis, each conferring an extended substrate spectrum. Single-amino-acid deletions T171del, I173del, and P174del and a two-amino-acid deletion, R165_T167delinsP, occurred in the omega loop, increasing the flexibility of the binding cavity. This rare collection of mutations has significance, allowing exploration of the diverse evolutionary trajectories of β-lactamases and as potential future mutations conferring high-level ceftazidime resistance on isolates from clinical settings, compared with amino acid substitution mutations.

Development of a random genomic DNA microarray for the detection and identification of Listeria monocytogenes in milk.

We developed a DNA microarray that contains random genomic DNA fragments of Listeria monocytogenes, validated its diagnostic abilities using cells grown in laboratory media and milk, and established enrichment conditions for detection of a low population of L. monocytogenes in milk. Genomic DNA of L.

Twelve positions in a β-lactamase that can expand its substrate spectrum with a single amino acid substitution.

The continuous evolution of β-lactamases resulting in bacterial resistance to β-lactam antibiotics is a major concern in public health, and yet the underlying molecular basis or the pattern of such evolution is largely unknown. We investigated the mechanics of the substrate fspectrum expansion of the class A β-lactamase using PenA of Burkholderia thailandensis as a model. By analyzing 516 mutated enzymes that acquired the ceftazidime-hydrolyzing activity, we found twelve positions with single amino acid substitutions (altogether twenty-nine different substitutions), co-localized at the active-site pocket area.

Substrate spectrum extension of PenA in Burkholderia thailandensis with a single amino acid deletion, Glu168del.

We describe a deletion mutation in a class A β-lactamase, PenA, of Burkholderia thailandensis that extended the substrate spectrum of the enzyme to include ceftazidime. Glu168del was located in a functional domain called the omega loop causing expansion of the space in the loop, which in turn increased flexibility at the active site. This deletion mutation represents a rare but significant alternative mechanical path to substrate spectrum extension in PenA besides more common substitution mutations.

The early stage of bacterial genome-reductive evolution in the host.

The equine-associated obligate pathogen Burkholderia mallei was developed by reductive evolution involving a substantial portion of the genome from Burkholderia pseudomallei, a free-living opportunistic pathogen. With its short history of divergence (approximately 3.5 myr), B.

Simple sequence repeat (SSR)-based gene diversity in Burkholderia pseudomallei and Burkholderia mallei.

Pathogens Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) contain a large number (> 12,000) of Simple Sequence Repeats (SSRs). To study the extent to which these features have contributed to the diversification of genes, we have conducted comparative studies with nineteen genomes of these bacteria. We found 210 genes with characteristic types of SSR variations.

Microarray data mining using landmark gene-guided clustering.

Background: Clustering is a popular data exploration technique widely used in microarray data analysis. Most conventional clustering algorithms, however, generate only one set of clusters independent of the biological context of the analysis. This is often inadequate to explore data from different biological perspectives and gain new insights.