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The Journal of Antimicrobial... Jul 2024The aac(6')-Im (aacA16) amikacin, netilmicin and tobramycin resistance gene cassette had been circulating globally undetected for many years in a sublineage of...
BACKGROUND
The aac(6')-Im (aacA16) amikacin, netilmicin and tobramycin resistance gene cassette had been circulating globally undetected for many years in a sublineage of Acinetobacter baumannii global clone 2.
OBJECTIVES
To identify sources for the aac(6')-Im fragment found in A. baumannii.
METHODS
MinION long-read sequencing and Unicycler hybrid assemblies were used to determine the genetic context of the aac(6')-Im gene. Quantitative reverse transcriptase PCR was used to measure expression.
RESULTS
Among >60 000 non-Acinetobacter draft genomes in the MRSN collection, the aac(6')-Im gene was detected in Pseudomonas putida and Enterobacter hormaechei isolates recovered from patients in Thailand between 2016 and 2019. Genomes of multiply resistant P. putida MRSN365855 and E. hormaechei MRSN791417 were completed. The class 1 integron containing the aac(6')-Im cassette was in the chromosome in MRSN365855, and in an HI2 plasmid in MRSN791417. However, MRSN791417 was amikacin susceptible and the gene was not expressed due to loss of the Pc promoter of the integron. Further examples of aac(6')-Im in plasmids from or the chromosome of various Gram-negative species were found in the GenBank nucleotide database. The aac(6')-Im context in integrons in pMRSN791417-8 and a Klebsiella plasmid pAMR200031 shared similarities with the aac(6')-Im region of AbGRI2-Im islands in A. baumannii. In other cases, the cassette array including the aac(6')-Im cassette was different.
CONCLUSIONS
The aac(6')-Im gene is widespread, being found so far in several different species and in several different gene cassette arrays. The lack of amikacin resistance in E. hormaechei highlights the importance of correlating resistance gene content and antibiotic resistance phenotype.
Topics: Anti-Bacterial Agents; Acinetobacter baumannii; Humans; Aminoglycosides; Microbial Sensitivity Tests; Drug Resistance, Multiple, Bacterial; Thailand; Integrons; Plasmids; Amikacin; Enterobacter; Bacterial Proteins; Tobramycin; Acetyltransferases; Genome, Bacterial
PubMed: 38742708
DOI: 10.1093/jac/dkae136 -
Bioresources and Bioprocessing May 2024Formolase (FLS) is a computationally designed enzyme that catalyzes the carboligation of two or three C1 formaldehyde molecules into C2 glycolaldehyde or C3...
BACKGROUND
Formolase (FLS) is a computationally designed enzyme that catalyzes the carboligation of two or three C1 formaldehyde molecules into C2 glycolaldehyde or C3 dihydroxyacetone (DHA). FLS lays the foundation for several artificial carbon fixation and valorization pathways, such as the artificial starch anabolic pathway. However, the application of FLS is limited by its low catalytic activity and product promiscuity.
FINDINGS
FLS, designed and engineered based on benzoylformate decarboxylase from Pseudomonas putida, was selected as a candidate for modification. To evaluate its catalytic activity, 25 residues located within an 8 Å distance from the active center were screened using single-point saturation mutagenesis. A screening approach based on the color reaction of the DHA product was applied to identify the desired FLS variants. After screening approximately 5,000 variants (approximately 200 transformants per site), several amino acid sites that were not identified by directed evolution were found to improve DHA formation. The serine-to-phenylalanine substitution at position 236 improved the activity towards DHA formation by 7.6-fold. Molecular dynamics simulations suggested that the mutation increased local hydrophobicity at the active site, predisposing the cofactor-C2 intermediate to nucleophilic attack by the third formaldehyde molecule for subsequent DHA generation.
CONCLUSIONS
This study provides improved FLS variants and valuable information into the influence of residues adjacent to the active center affecting catalytic efficiency, which can guide the rational engineering or directed evolution of FLS to optimize its performance in artificial carbon fixation and valorization.
PubMed: 38735884
DOI: 10.1186/s40643-024-00767-3 -
International Journal of Biological... Jun 2024Polycyclic aromatic hydrocarbons (PAHs) are molecules with two or more fused aromatic rings that occur naturally in the environment due to incomplete combustion of...
Polycyclic aromatic hydrocarbons (PAHs) are molecules with two or more fused aromatic rings that occur naturally in the environment due to incomplete combustion of organic substances. However, the increased demand for fossil fuels in recent years has increased anthropogenic activity, contributing to the environmental concentration of PAHs. The enzyme chlorocatechol 1,2-dioxygenase from Pseudomonas putida (Pp 1,2-CCD) is responsible for the breakdown of the aromatic ring of catechol, making it a potential player in bioremediation strategies. Pp 1,2-CCD can tolerate a broader range of substrates, including halogenated compounds, than other dioxygenases. Here, we report the construction of a chimera protein able to form biomolecular condensates with potential application in bioremediation. The chimera protein was built by conjugating Pp 1,2-CCD to low complex domains (LCDs) derived from the DEAD-box protein Dhh1. We showed that the chimera could undergo liquid-liquid phase separation (LLPS), forming a protein-rich liquid droplet under different conditions (variable protein and PEG8000 concentrations and pH values), in which the protein maintained its structure and main biophysical properties. The condensates were active against 4-chlorocatechol, showing that the chimera droplets preserved the enzymatic activity of the native protein. Therefore, it constitutes a prototype of a microreactor with potential use in bioremediation.
Topics: Dioxygenases; Polycyclic Aromatic Hydrocarbons; Biodegradation, Environmental; Pseudomonas putida; Catechols; Bacterial Proteins
PubMed: 38735602
DOI: 10.1016/j.ijbiomac.2024.132294 -
Microbial Pathogenesis Jul 2024The emergence of carbapenem-resistant Pseudomonas putida (CRPP) has raised public awareness. This study investigated two strains from the Pseudomonas putida group that...
OBJECTIVES
The emergence of carbapenem-resistant Pseudomonas putida (CRPP) has raised public awareness. This study investigated two strains from the Pseudomonas putida group that were resistant to carbapenem, tigecycline, and aztreonam-avibactam (ATM-AVI), with a focus on their microbial and genomic characteristics.
METHODS
We assessed the antibiotic resistance profile using broth dilution, disk diffusion, and E-test methods. Efflux pump phenotype testing and real-time quantitative PCR were employed to evaluate efflux pump activity in tigecycline resistance, while polymerase chain reaction was utilized to detect common carbapenem genes. Additionally, whole-genome sequencing was performed to analyze genomic characteristics. The transferability of bla and bla was assessed through a conjugation experiment. Furthermore, growth kinetics and biofilm formation were examined using growth curves and crystal violet staining.
RESULTS
Both strains demonstrated resistance to carbapenem, tigecycline, and ATM-AVI. Notably, NMP can restore sensitivity to tigecycline. Subsequent analysis revealed that they co-produced bla, bla, tmexCD-toprJ, and bla, belonging to a novel sequence type ST268. Although they were closely related on the phylogenetic tree, they exhibited different levels of virulence. Genetic environment analysis indicated variations compared to prior studies, particularly regarding the bla and bla genes, which showed limited horizontal transferability. Moreover, it was observed that temperature exerted a specific influence on their biological factors.
CONCLUSION
We initially identified two P. putida ST268 strains co-producing bla, bla, bla, and tmexCD-toprJ. The resistance to tigecycline and ATM-AVI can be attributed to the presence of multiple drug resistance determinants. These findings underscore the significance of P. putida as a reservoir for novel antibiotic resistance genes. Therefore, it is imperative to develop alternative antibiotic therapies and establish effective monitoring of bacterial resistance.
Topics: Pseudomonas putida; Tigecycline; Anti-Bacterial Agents; Microbial Sensitivity Tests; China; Aztreonam; Azabicyclo Compounds; beta-Lactamases; Drug Resistance, Multiple, Bacterial; Whole Genome Sequencing; Humans; Drug Combinations; Biofilms; Bacterial Proteins; Pseudomonas Infections; Carbapenems
PubMed: 38697232
DOI: 10.1016/j.micpath.2024.106668 -
Frontiers in Microbiology 2024Pholiota nameko is a widely consumed edible fungus. This study focuses on two crucial developmental stages of Pholiota nameko, namely, mycelium and ascospores. The...
INTRODUCTION
Pholiota nameko is a widely consumed edible fungus. This study focuses on two crucial developmental stages of Pholiota nameko, namely, mycelium and ascospores. The objectives of this research were to investigate changes in microbial diversity and community structure during the growth of Pholiota nameko and to analyze the adaptability of the dominant strains to their respective habitats through metabolic.
METHODS
Specifically, we conducted second-generation sequencing of the 16S rRNA gene (Illumina) on samples obtained from these stages. In addition, we isolated and characterized endophytes present in Pholiota nameko, focusing on examining the impact of dominant endophyte genera on autolysis. We also conducted a metabolic pathway analysis.
RESULTS AND DISCUSSION
The results unveiled 578,414 valid sequences of Pholiota nameko endophytic fungi. At the phylum level, the dominant taxa were Basidiomycota, Ascomycota, Zoopagomycota, and Mucoromycota. At the genus level, the dominant taxa observed were Pholiota, Inocybe, Fusarium, and Hortiboletus. For endophytic bacteria, we obtained 458,475 valid sequences. The dominant phyla were Proteobacteria, TM6, Firmicutes, and Bacteroidetes, while the dominant genera were Edaphobacter, Xanthomonas, Burkholderia, and Pseudomonas. Moreover, we identified the isolated strains in Pholiota nameko using 16S rDNA, and most of them were found to belong to the genus Pseudomonas, with Pseudomonas putida being the most prevalent strain. The findings revealed that the Pseudomonas putida strain has the ability to slow down the breakdown of soluble proteins and partially suppress the metabolic processes that generate superoxide anion radicals in Pholiota nameko, thereby reducing autolysis. Additionally, our results demonstrated that molybdenum enzyme-mediated anaerobic oxidative phosphorylation reactions were the primary energy metabolism pathway in the Pseudomonas putida strain. This suggests that the molybdenum cofactor synthesis pathway might be the main mechanism through which Pholiota nameko adapts to its complex and diverse habitats.
PubMed: 38690362
DOI: 10.3389/fmicb.2024.1319886 -
Pharmaceutics Apr 2024Dasatinib (DAS), a potent anticancer drug, has been subjected to formulation enhancements due to challenges such as significant first-pass metabolism, poor absorption,...
Dasatinib (DAS), a potent anticancer drug, has been subjected to formulation enhancements due to challenges such as significant first-pass metabolism, poor absorption, and limited oral bioavailability. To improve its release profile, DAS was embedded in a matrix of the hydrophilic polymer polyvinylpyrrolidone (PVP). Drug amorphization was induced in a planetary ball mill by solvent-free co-grinding, facilitating mechanochemical activation. This process resulted in the formation of amorphous solid dispersions (ASDs). The ASD capsules exhibited a notable enhancement in the release rate of DAS compared to capsules containing the initial drug. Given that anticancer drugs often undergo limited metabolism in the body with unchanged excretion, the ecotoxicological effect of the native form of DAS was investigated as well, considering its potential accumulation in the environment. The highest ecotoxicological effect was observed on the bacteria , while other test organisms (bacteria , microalgae sp., and duckweed ) exhibited negligible effects. The enhanced drug release not only contributes to improved oral absorption but also has the potential to reduce the proportion of DAS that enters the environment through human excretion. This comprehensive approach highlights the significance of integrating advances in drug development while considering its environmental implications.
PubMed: 38675212
DOI: 10.3390/pharmaceutics16040551 -
Microorganisms Apr 2024Avocado is one of the most in-demand fruits worldwide and the trend towards its sustainable production, regulated by international standards, is increasing. One of the...
Avocado is one of the most in-demand fruits worldwide and the trend towards its sustainable production, regulated by international standards, is increasing. One of the most economically important diseases is root rot, caused by . Regarding this problem, antagonistic microorganism use is an interesting alternative due to their phytopathogen control efficiency. Therefore, the interaction of arbuscular mycorrhizal fungi of the phylum Glomeromycota, native to the Peruvian coast (GWI) and jungle (GFI), and avocado rhizospheric bacteria, and , was evaluated in terms of their biocontrol capacity against in the "Zutano" variety of avocado plants. The results showed that the GWI and combination increased the root exploration surface by 466.36%. increased aerial biomass by 360.44% and increased root biomass by 433.85%. Likewise, rhizobacteria showed the highest nitrogen (24.60 mg ∙ g DM) and sulfur (2.60 mg ∙ g DM) concentrations at a foliar level. The combination of GWI and was the treatment that presented the highest calcium (16.00 mg ∙ g DM) and magnesium (8.80 mg ∙ g DM) concentrations. The microorganisms' multifunctionality reduced disease severity by 85 to 90% due to the interaction between mycorrhizae and rhizobacteria. In conclusion, the use of growth promoting microorganisms that are antagonistic to represents a potential strategy for sustainable management of avocado cultivation.
PubMed: 38674665
DOI: 10.3390/microorganisms12040721 -
International Journal of Molecular... Apr 2024The potato dry rot disease caused by spp. seriously reduces potato yield and threatens human health. However, potential biocontrol agents cannot guarantee the stability...
The potato dry rot disease caused by spp. seriously reduces potato yield and threatens human health. However, potential biocontrol agents cannot guarantee the stability and activity of biocontrol. Here, 18 synthetic microbial communities of different scales were constructed, and the synthetic microbial communities with the best biocontrol effect on potato dry rot disease were screened through in vitro and in vivo experiments. The results show that the synthetic community composed of , , , , and has the best biocontrol activity. Metabolomics results show that interacts with other member strains to produce caproic acid and reduce the disease index to 38.01%. Furthermore, the mycelial growth inhibition after treatment with caproic acid was 77.54%, and flow cytometry analysis showed that the living conidia rate after treatment with caproic acid was 11.2%. This study provides potential value for the application of synthetic microbial communities in potatoes, as well as the interaction mechanisms between member strains of synthetic microbial communities.
Topics: Solanum tuberosum; Plant Diseases; Biological Control Agents; Fusarium; Microbial Interactions; Bacteria; Microbial Consortia
PubMed: 38674022
DOI: 10.3390/ijms25084437 -
Frontiers in Bioengineering and... 2024Polyhydroxyalkanoates (PHAs) are biodegradable polymers that can be produced from lignocellulosic biomass by microorganisms. Cheap and readily available raw material,...
Polyhydroxyalkanoates (PHAs) are biodegradable polymers that can be produced from lignocellulosic biomass by microorganisms. Cheap and readily available raw material, such as corn stover waste, has the potential to lessen the cost of PHA synthesis. In this research study, corn stover is pretreated with NaOH under conditions optimized for high cellulose and low lignin with central composite design (CCD) followed by characterization using Fourier-transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), and scanning electron microscopy (SEM). Design expert software performed further optimization of alkali pretreated corn stover for high total reducing sugar (TRS) enhancement using CCD using response surface methodology (RSM). The optimized condition by RSM produced a TRS yield of 707.19 mg/g. Fermentation using corn stover hydrolysate by MTCC 2475 gave mcl-PHA detected through g (GC-MS/MS) and characterization of the PHA film by differential scanning calorimetry (DSC), FTIR, and nuclear magnetic resonance (NMR). Thus, this research paper focuses on using agriculture (stubble) waste as an alternative feedstock for PHA production.
PubMed: 38665813
DOI: 10.3389/fbioe.2024.1343579 -
BMC Veterinary Research Apr 2024The present study aimed to determine the major cause of the high mortality affecting farmed gilthead seabream (Sparus aurata) and controlling this disease condition....
The present study aimed to determine the major cause of the high mortality affecting farmed gilthead seabream (Sparus aurata) and controlling this disease condition. Fifteen diseased S. aurata were sampled from a private fish farm located at Eldeba Triangle, Damietta, fish showed external skin hemorrhages, and ulceration. Bacterial isolates retrieved from the diseased fish were identified biochemically as Pseudomonas putida and then confirmed by phylogenetic analysis of the 16 S rRNA gene sequence. P. putida was also isolated from three batches of tilapia-trash feed given to S. aurata. Biofilm and hemolytic assay indicated that all P. putida isolates produced biofilm, but 61.11% can haemolyse red blood cells. Based on the antibiotic susceptibility test results, P. putida was sensitive to florfenicol with minimum inhibitory concentrations ranging between 0.25 and 1.0 µg mL, but all isolates were resistant to ampicillin and sulfamethoxazole-trimethoprim. Pathogenicity test revealed that P. putida isolate (recovered from the tilapia-trash feed) was virulent for S. aurata with LD equal to 4.67 × 10 colony forming unit (CFU) fish. After intraperitoneal (IP) challenge, fish treated with 10 mg kg of florfenicol showed 16.7% mortality, while no mortality was recorded for the fish group that received 20 mg kg. The non-treated fish group showed 46.7% mortality after bacterial challenge. HPLC analysis of serum florfenicol levels reached 1.07 and 2.52 µg mL at the 5th -day post-drug administration in the fish groups received 10 and 20 mg kg, respectively. In conclusion, P. putida was responsible for the high mortality affecting cultured S. aurata, in-feed administration of florfenicol (20 mg kg) effectively protected the challenged fish.
Topics: Animals; Thiamphenicol; Fish Diseases; Pseudomonas putida; Anti-Bacterial Agents; Animal Feed; Sea Bream; Pseudomonas Infections; Microbial Sensitivity Tests; Tilapia; Phylogeny; RNA, Ribosomal, 16S; Biofilms
PubMed: 38664683
DOI: 10.1186/s12917-024-04004-z