Pharmacokinetics and safety evaluation of intravenously administered Pseudomonas phage PA_LZ7 in a mouse model.

IMPORTANCE: Phage therapy is gaining traction as an alternative to antibiotics due to the rise of multi-drug-resistant (MDR) bacteria. This study assessed the pharmacokinetics and safety of PA_LZ7, a phage targeting MDR Pseudomonas aeruginosa, in mice. After intravenous administration, the phage showed an exponential decay in plasma and its concentration dropped significantly within 24 h for all dosage groups. Although there was a temporary increase in certain plasma cytokines and spleen weight at higher dosages, no significant toxicity was observed. Therefore, PA_LZ7 shows potential as an effective and safe candidate for future phage therapy against MDR P. aeruginosa infections.

Engineered phage with cell-penetrating peptides for intracellular bacterial infections.

IMPORTANCE: Salmonella infection is a significant threat to global public health, and the increasing prevalence of antibiotic resistance exacerbates the situation. Therefore, finding new and effective ways to combat this pathogen is essential. Phages are natural predators of bacteria and can be used as an alternative to antibiotics to kill specific bacteria, including drug-resistant strains. One significant limitation of using phages as antimicrobial agents is their low cellular uptake, which limits their effectiveness against intracellular bacterial infections. Therefore, finding ways to enhance phage uptake is crucial. Our study provides a straightforward strategy for displaying cell-penetrating peptides on non-model phages, offering a promising novel and effective therapeutic approach for treating intracellular and drug-resistant bacteria. This approach has the potential to address the global challenge of antibiotic resistance and improve public health outcomes.

Mucilaginibacter xinganensis sp. nov., a phenanthrene-degrading bacterium isolated from wetland soil.

An aerobic, Gram-stain negative, rod-shaped and non-motile strain, BJC16-A31T, was isolated from the wetland soil sample taken from Daxing'anling, Heilongjiang, People's Republic of China. Strain BJC16-A31T was found to be oxidase- and catalase-positive, and produced light orange colonies on modified R2A agar. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain BJC16-A31T is closely related to Mucilaginibacter gotjawali SA3-7T with 96.54% sequence similarity and it formed a separate lineage in the genus Mucilaginibacter. Strain BJC16-A31T contained menaquinone-7 (MK-7) as the predominant isoprenoid quinine. Anteiso-C15:0, C16:0 and anteiso-C15:0 were the major fatty acids. The major polar lipids were phosphatidylethanolamine, six unidentified polar lipid, two unidentified aminophospholipids and one unidentified aminolipid. The genome is composed of a circular 5,301,339 bp chromosome with average G + C percentage of 42.25%. The Average Nucleotide Identity (ANI) between strain BJC16-A31T and M. gotjawali SA3-7T was 77.51%. Combined phenotypic, chemotaxonomic, phylogenetic and genomic characteristics support the conclusion that strain BJC16-A31T represents a novel species of the genus Mucilaginibacter, for which the name Mucilaginibacter xinganensis sp. nov. is proposed. The type strain is BJC16-A31T (= CGMCC 1.12728T = NBRC 110384T).

The complete genome of Comamonas testosteroni reveals its genetic adaptations to changing environments.

Members of the gram-negative, strictly aerobic genus Comamonas occur in various environments. Here we report the complete genome of Comamonas testosteroni strain CNB-2. Strain CNB-2 has a circular chromosome that is 5,373,643 bp long and has a G+C content of 61.4%. A total of 4,803 open reading frames (ORFs) were identified; 3,514 of these ORFs are functionally assigned to energy production, cell growth, signal transduction, or transportation, while 866 ORFs encode hypothetical proteins and 423 ORFs encode purely hypothetical proteins. The CNB-2 genome has many genes for transportation (22%) and signal transduction (6%), which allows the cells to respond and adapt to changing environments. Strain CNB-2 does not assimilate carbohydrates due to the lack of genes encoding proteins involved in glycolysis and pentose phosphate pathways, and it contains many genes encoding proteins involved in degradation of aromatic compounds. We identified 66 Tct and nine TRAP-T systems and a complete tricarboxylic acid cycle, which may allow CNB-2 to take up and metabolize a range of carboxylic acids. This nutritional bias for carboxylic acids and aromatic compounds enables strain CNB-2 to occupy unique niches in environments. Four different sets of terminal oxidases for the respiratory system were identified, and they putatively functioned at different oxygen concentrations. This study conclusively revealed at the genomic level that the genetic versatility of C. testosteroni is vital for competition with other bacteria in its special niches.

Responses to arsenate stress by Comamonas sp. strain CNB-1 at genetic and proteomic levels.

Comamonas sp. strain CNB-1, a chloronitrobenzene-degrading bacterium, was demonstrated to possess higher arsenate tolerance as compared with the mutant strain CNB-2. pCNB1, a plasmid harboured by CNB-1 but not CNB-2, contained the genetic cluster ars(RPBC)Com, which putatively encodes arsenate-resistance regulator, family II arsenate reductase, arsenite efflux pump and family I arsenate reductase, respectively, in Comamonas strain CNB-1. The arsC-negative Escherichia coli could gain arsenate resistance by transformation with arsPCom or arsCCom, indicating that these two genes might express functional forms of arsenate reductases. Intriguingly, when CNB-1 cells were exposed to arsenate, the transcription of arsPCom and arsCCom was measurable by RT-PCR, but only ArsPCom was detectable at protein level. To explore the proteins responding to arsenate stress, CNB-1 cells were cultured with and without arsenate and differential proteomics was carried out by two-dimensional PAGE (2-DE) and MALDI-TOF MS. A total of 31 differential 2-DE spots were defined upon image analysis and 23 proteins were identified to be responsive specifically to arsenate. Of these spots, 18 were unique proteins. These proteins were identified to be phosphate transporters, heat-shock proteins involved in protein refolding, and enzymes participating in carbon and energy metabolism.

Nucleotide sequence of plasmid pCNB1 from comamonas strain CNB-1 reveals novel genetic organization and evolution for 4-chloronitrobenzene degradation.

The nucleotide sequence of a new plasmid pCNB1 from Comamonas sp. strain CNB-1 that degrades 4-chloronitrobenzene (4CNB) was determined. pCNB1 belongs to the IncP-1beta group and is 91,181 bp in length. A total of 95 open reading frames appear to be involved in (i) the replication, maintenance, and transfer of pCNB1; (ii) resistance to arsenate and chromate; and (iii) the degradation of 4CNB. The 4CNB degradative genes and arsenate resistance genes were located on an extraordinarily large transposon (44.5 kb), proposed as TnCNB1. TnCNB1 was flanked by two IS1071 elements and represents a new member of the composite I transposon family. The 4CNB degradative genes within TnCNB1 were separated by various truncated genes and genetic homologs from other DNA molecules. Genes for chromate resistance were located on another transposon that was similar to the Tn21 transposon of the class II replicative family that is frequently responsible for the mobilization of mercury resistance genes. Resistance to arsenate and chromate were experimentally confirmed, and transcriptions of arsenate and chromate resistance genes were demonstrated by reverse transcription-PCR. These results described a new member of the IncP-1beta plasmid family, and the findings suggest that gene deletion and acquisition as well as genetic rearrangement of DNA molecules happened during the evolution of the 4CNB degradation pathway on pCNB1.

A novel deaminase involved in chloronitrobenzene and nitrobenzene degradation with Comamonas sp. strain CNB-1.

Comamonas sp. strain CNB-1 degrades nitrobenzene and chloronitrobenzene via the intermediates 2-aminomuconate and 2-amino-5-chloromuconate, respectively. Deamination of these two compounds results in the release of ammonia, which is used as a source of nitrogen for bacterial growth. In this study, a novel deaminase was purified from Comamonas strain CNB-1, and the gene (cnbZ) encoding this enzyme was cloned. The N-terminal sequence and peptide fingerprints of this deaminase were determined, and BLAST searches revealed no match with significant similarity to any functionally characterized proteins. The purified deaminase is a monomer (30 kDa), and its V(max) values for 2-aminomuconate and 2-amino-5-chloromuconate were 147 micromol x min(-1) x mg(-1) and 196 micromol x min(-1) x mg(-1), respectively. Its catalytic products from 2-aminomuconate and 2-amino-5-chloromuconate were 2-hydroxymuconate and 2-hydroxy-5-chloromuconate, respectively, which are different from those previously reported for the deaminases of Pseudomonas species. In the catalytic mechanism proposed, the alpha-carbon and nitrogen atoms (of both 2-aminomuconate and 2-amino-5-chloromuconate) were simultaneously attacked by a hydroxyl group and a proton, respectively. Homologs of cnbZ were identified in the genomes of Bradyrhizobium japonicum, Rhodopseudomonas palustris, and Roseiflexus sp. strain RS-1; these genes were previously annotated as encoding hypothetical proteins of unknown function. It is concluded that CnbZ represents a novel enzyme that deaminates xenobiotic compounds and/or alpha-amino acids.

Novel partial reductive pathway for 4-chloronitrobenzene and nitrobenzene degradation in Comamonas sp. strain CNB-1.

Comamonas sp. strain CNB-1 grows on 4-chloronitrobenzene (4-CNB) and nitrobenzene as sole carbon and nitrogen sources. In this study, two genetic segments, cnbB-orf2-cnbA and cnbR-orf1-cnbCaCbDEFGHI, located on a newly isolated plasmid, pCNB1 (ca. 89 kb), and involved in 4-CNB/nitrobenzene degradation, were characterized. Seven genes (cnbA, cnbB, cnbCa, cnbCb, cnbD, cnbG, and cnbH) were cloned and functionally expressed in recombinant Escherichia coli, and they were identified as encoding 4-CNB nitroreductase (CnbA), 1-hydroxylaminobenzene mutase (CnbB), 2-aminophenol 1,6-dioxygenase (CnbCab), 2-amino-5-chloromuconic semialdehyde dehydrogenase (CnbD), 2-hydroxy-5-chloromuconic acid (2H5CM) tautomerase, and 2-amino-5-chloromuconic acid (2A5CM) deaminase (CnbH). In particular, the 2A5CM deaminase showed significant identities (31 to 38%) to subunit A of Asp-tRNAAsn/Glu-tRNAGln amidotransferase and not to the previously identified deaminases for nitroaromatic compound degradation. Genetic cloning and expression of cnbH in Escherichia coli revealed that CnbH catalyzed the conversion of 2A5CM into 2H5CM and ammonium. Four other genes (cnbR, cnbE, cnbF, and cnbI) were tentatively identified according to their high sequence identities to other functionally identified genes. It was proposed that CnbH might represent a novel type of deaminase and be involved in a novel partial reductive pathway for chloronitrobenzene or nitrobenzene degradation.

[Isolation and identification of two marine bacteria with hydrocarbon-biodegradation activity].

Two bacterial strains, able to degrade both diesel fuel and PAHs, were isolated from the oil sewage of oil storage dock in Xiamen. Analysis of 16S rDNA sequences showed that both strains had high homology (99%) with Pseudomonas stutzeri and should be classified to this species. But Biolog analysis showed they were quite different strains. Tests of temperature, salinity, pH on cell growth suggested that they were from marine environment. Both showed high degradation activity to naphthalene, 87.53% and 84.01% within 3 days; and slight activity to pyrene, 8.35% and 5.37% within 7 days. And both harbored naphthalene dioxygenase genes of 98% identity with other those from Pseudomonas strains.