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MicrobiologyOpen Oct 2023Liquid soap dispensers are widely used in domestic and clinical settings. In previous studies, the risk of bacterial contamination of refillable systems was pointed out...
Liquid soap dispensers are widely used in domestic and clinical settings. In previous studies, the risk of bacterial contamination of refillable systems was pointed out and a bacterial contamination rate of 25%, with values of up to 10 colony-forming units/mL (CFU/mL), was reported. However, the route of contamination remains elusive. To address this point, we determined the microbial contamination of refillable standard pump dispensers and nonrefillable press-dispenser systems. Following the collection of 104 liquid soap dispensers from hotel rooms across Germany, bacterial counts were determined. Isolates of samples containing nonfastidious Gram-negative bacteria were further analyzed by the Vitek 2 system for the determination of species. 70.2% of the refillable pump dispensers (mean total bacterial count = 2.2 × 10 CFU/mL) but only 10.6% of the nonrefillable press dispensers, were contaminated (mean total bacterial count = 1.5 × 10 CFU/mL). Of samples containing nonfastidious Gram-negative bacteria, Pluralibacter gergoviae was present in 41.7%, Pseudomonads (Pseudomonas aeruginosa and Pseudomonas putida) in 25%, Serratia marcescens in 16.7%, and Klebsiella oxytoca and Pasteurella testudinis in 8.3%. After the initial assessment, we contaminated different dispensing systems with P. aeruginosa/P. gergoviae, to reveal the route of contamination and identied the pressure release of standard pump dispensers as the loophole for microbial contamination.
Topics: Soaps; Gram-Negative Bacteria; Bacterial Load; Serratia marcescens; Germany
PubMed: 37877653
DOI: 10.1002/mbo3.1384 -
Synthetic and Systems Biotechnology Sep 2023Industrial expansion has led to environmental pollution by xenobiotic compounds like polycyclic aromatic hydrocarbons and monoaromatic hydrocarbons. spp. have broad...
Industrial expansion has led to environmental pollution by xenobiotic compounds like polycyclic aromatic hydrocarbons and monoaromatic hydrocarbons. spp. have broad metabolic potential for degrading aromatic compounds. The objective of this study was to develop a "biological funneling" strategy based on genetic modification to convert complex aromatic compounds into -muconate (ccMA) using B6-2 and MPDS as biocatalysts. The engineered strains B6-2 (B6-2ΔcatBΔsalC) and MPDS (MPDSΔsalC(pUCP18k-catA)) thrived with biphenyl or naphthalene as the sole carbon source and produced ccMA, attaining molar conversions of 95.3% (ccMA/biphenyl) and 100% (ccMA/naphthalene). Under mixed substrates, B6-2ΔcatBΔsalC grew on biphenyl as a carbon source and transformed ccMA from non-growth substrates benzoate or salicylate to obtain higher product concentration. Inserting exogenous clusters like and allowed B6-2 recombinant strains to convert benzene and toluene to ccMA. In mixed substrates, constructed consortia of engineered strains B6-2 and MPDS specialized in catabolism of biphenyl and naphthalene; the highest molar conversion rate of ccMA from mixed substrates was 85.2% when B6-2ΔcatBΔsalC was added after 24 h of MPDSΔsalC(pUCP18k-catA) incubation with biphenyl and naphthalene. This study provides worthwhile insights into efficient production of ccMA from aromatic hydrocarbons by reusing complex pollutants.
PubMed: 37637202
DOI: 10.1016/j.synbio.2023.08.001 -
Journal of Microbiology and... Aug 2023(formerly ) is Gram-positive, and spore-forming bacteria causing food spoilage, especially in acidic canned food products. To control , we isolated a bacteriophage...
(formerly ) is Gram-positive, and spore-forming bacteria causing food spoilage, especially in acidic canned food products. To control , we isolated a bacteriophage Youna2 from a sewage sludge sample. Morphological analysis revealed that phage Youna2 belongs to the family with a non-contractile and flexible tail. Youna2 has 52,903 bp double-stranded DNA containing 61 open reading frames. There are no lysogeny-related genes, suggesting that Youna2 is a virulent phage. , a putative endolysin gene was identified in the genome of Youna2 and predicted to be composed of a -acetylmuramoyl-L-alanine amidase domain (PF01520) at the N-terminus and unknown function DUF5776 domain (PF19087) at the C-terminus. While phage Youna2 has a narrow host range, infecting only certain strains of , PlyYouna2 exhibited a broad antimicrobial spectrum beyond the genus. Interestingly, PlyYouna2 can lyse Gram-negative bacteria such as , , and without other additives to destabilize bacterial outer membrane. To the best of our knowledge, Youna2 is the first -infecting phage and we speculate its endolysin PlyYouna2 can provide the basis for the development of a novel biocontrol agent against various foodborne pathogens.
Topics: Bacteriophages; Bacillus coagulans; Endopeptidases; Siphoviridae; Genome, Viral
PubMed: 37218442
DOI: 10.4014/jmb.2303.03021 -
Infection, Disease & Health Nov 2023Bacterial contamination on inanimate clinical surfaces is directly linked to severe health problems, especially those caused by multidrug resistant (MDR) pathogens....
BACKGROUND
Bacterial contamination on inanimate clinical surfaces is directly linked to severe health problems, especially those caused by multidrug resistant (MDR) pathogens. Here, we evaluated the microbial burden in these environments and tested the efficacy of a novel HLE disinfectant solution.
METHODS
Microbial contamination of healthcare surfaces [Intensive Care Unit (ICU), Long Period Hospitalization Room (LPHR) and Otolaryngology Consultation (OC)] and the efficacy of HLE disinfectant solution were determined analyzing the viable counts on general and selective media, and also by molecular studies focused on metagenomic and specific qPCR.
RESULTS
Different contamination loads were detected with LPHR showing the highest contamination. Treatment with the HLE disinfectant solution curbed the spread of well-adapted pathogens on touched surfaces (ICU, LPHR and OC). Metagenomic analysis of microbial diversity of the Patient Table (most contaminated surface in LPHR) revealed the presence of mainly A. johnsonii and P. putida. Furthermore, functional annotation of toxin, virulence and antibiotic resistance sequences showed a high diversity of Acinetobacter spp. and Pseudomonas spp. In this context, specific qPCR analysis confirmed the efficacy of HLE disinfectant solution against the most prevalent and critical pathogens Pseudomonas sp. and Acinetobacter sp. achieving their complete eradication.
CONCLUSION
Given the persistence of detrimental resistant pathogens, the application of HLE disinfection solution could be a highly beneficial and effective option -used either alone or in combination-for infection prevention and control with the aim to eliminate microbial pathogens and their genes from contaminated contact-surfaces and thus limit the spread to humans and other ecological niches.
Topics: Humans; Disinfectants; Disinfection; Bacteria; Drug Resistance, Microbial; Delivery of Health Care
PubMed: 37558566
DOI: 10.1016/j.idh.2023.07.002 -
Microbial Biotechnology Jan 2024Providing an anodic potential in a bio-electrochemical system to the obligate aerobe Pseudomonas putida enables anaerobic survival and allows the cells to overcome redox...
Providing an anodic potential in a bio-electrochemical system to the obligate aerobe Pseudomonas putida enables anaerobic survival and allows the cells to overcome redox imbalances. In this setup, the bacteria could be exploited to produce chemicals via oxidative pathways at high yield. However, the absence of anaerobic growth and low carbon turnover rates remain as obstacles for the application of such an electro-fermentation technology. Growth and carbon turnover start with carbon uptake into the periplasm and cytosol. P. putida KT2440 has three native transporting systems for glucose, each differing in energy and redox demand. This architecture previously led to the hypothesis that internal redox and energy constraints ultimately limit cytoplasmic carbon utilization in a bio-electrochemical system. However, it remains largely unclear which uptake route is predominantly used by P. putida under electro-fermentative conditions. To elucidate this, we created three gene deletion mutants of P. putida KT2440, forcing the cells to exclusively utilize one of the routes. When grown in a bio-electrochemical system, the pathway mutants were heavily affected in terms of sugar consumption, current output and product formation. Surprisingly, however, we found that about half of the acetate formed in the cytoplasm originated from carbon that was put into the system via the inoculation biomass, while the other half came from the consumption of substrate. The deletion of individual sugar uptake routes did not alter significantly the secreted acetate concentrations among different strains even with different carbon sources. This means that the stoichiometry of the sugar uptake routes is not a limiting factor during electro-fermentation and that the low rates might be caused by other reasons, for example energy limitations or a yet-to-be-identified oxygen-dependent regulatory mechanism.
Topics: Pseudomonas putida; Anaerobiosis; Glucose; Carbon; Acetates
PubMed: 37990843
DOI: 10.1111/1751-7915.14375 -
ACS Synthetic Biology Jul 2023It is impractical to develop a new parts collection for every potential host organism. It is well-established that gene expression parts, like genes, are qualitatively...
It is impractical to develop a new parts collection for every potential host organism. It is well-established that gene expression parts, like genes, are qualitatively transferable, but there is little quantitative information defining transferability. Here, we systematically quantified the behavior of a parts set across multiple hosts. To do this, we developed a broad host range (BHR) plasmid system compatible with the large, modular CIDAR parts collection for , which we named openCIDAR. This enabled testing of a library of DNA constructs across the Pseudomonadota─, , , and . Part performance was evaluated with a standardized characterization procedure that quantified expression in terms of molecules of equivalent fluorescein (MEFL), an objective unit of measure. The results showed that the CIDAR parts enable graded gene expression across all organisms─meaning that the same parts can be used to program , , , and . Most parts had a similar expression trend across hosts, although each organism had a different average gene expression level. The variability is enough that to achieve the same MEFL in a different organism, a lookup table is required to translate a design from one host to another. To identify truly divergent parts, we applied linear regression to a combinatorial set of promoters and ribosome binding sites, finding that the promoter J23100 behaves very differently in than in the other hosts. Thus, it is now possible to evaluate any CIDAR compatible part in three other hosts of interest, and the diversity of these hosts implies that the collection will also be compatible with many other Proteobacteria (Pseudomonadota). Furthermore, this work defines an approach to generalize modular synthetic biology parts sets beyond a single host, implying that only a few parts sets may be needed to span the tree of life. This will accelerate current efforts to engineer diverse species for environmental, biotechnological, and health applications.
Topics: Escherichia coli; Gene Library; Biotechnology; Promoter Regions, Genetic; Plasmids
PubMed: 37294017
DOI: 10.1021/acssynbio.3c00104 -
Bioelectrochemistry (Amsterdam,... Jun 2024Pseudomonas aeruginosa phenazines contribute to survival under microaerobic and anaerobic conditions by extracellular electron discharge to regulate cellular redox...
Pseudomonas aeruginosa phenazines contribute to survival under microaerobic and anaerobic conditions by extracellular electron discharge to regulate cellular redox balances. This electron discharge is also attractive to be used for bioelectrochemical applications. However, elements of the respiratory pathways that interact with phenazines are not well understood. Five terminal oxidases are involved in the aerobic electron transport chain (ETC) of Pseudomonas putida and P. aeruginosa. The latter bacterium also includes four reductases that allow for denitrification. Here, we explored if phenazine-1-carboxylic acid interacts with those elements to enhance anodic electron discharge and drive bacterial growth in oxygen-limited conditions. Bioelectrochemical evaluations of terminal oxidase-deficient mutants of both Pseudomonas strains and P. aeruginosa with stimulated denitrification pathways indicated no direct beneficial interaction of phenazines with ETC elements for extracellular electron discharge. However, the single usage of the Cbb3-2 oxidase increased phenazine production, electron discharge, and cell growth. Assays with purified periplasmic cytochromes NirM and NirS indicated that pyocyanin acts as their electron donor. We conclude that phenazines play an important role in electron transfer to, between, and from terminal oxidases under oxygen-limiting conditions and their modulation might enhance EET. However, the phenazine-anode interaction cannot replace oxygen respiration to deliver energy for biomass formation.
Topics: Electron Transport; Pseudomonas aeruginosa; Pseudomonas putida; Electrons; Phenazines; Oxygen
PubMed: 38181591
DOI: 10.1016/j.bioelechem.2023.108636 -
Metabolic Engineering Jun 2024Sunscreen has been used for thousands of years to protect skin from ultraviolet radiation. However, the use of modern commercial sunscreen containing oxybenzone, ZnO,...
Sunscreen has been used for thousands of years to protect skin from ultraviolet radiation. However, the use of modern commercial sunscreen containing oxybenzone, ZnO, and TiO has raised concerns due to their negative effects on human health and the environment. In this study, we aim to establish an efficient microbial platform for production of shinorine, a UV light absorbing compound with anti-aging properties. First, we methodically selected an appropriate host for shinorine production by analyzing central carbon flux distribution data from prior studies alongside predictions from genome-scale metabolic models (GEMs). We enhanced shinorine productivity through CRISPRi-mediated downregulation and utilized shotgun proteomics to pinpoint potential competing pathways. Simultaneously, we improved the shinorine biosynthetic pathway by refining its design, optimizing promoter usage, and altering the strength of ribosome binding sites. Finally, we conducted amino acid feeding experiments under various conditions to identify the key limiting factors in shinorine production. The study combines meta-analysis of C-metabolic flux analysis, GEMs, synthetic biology, CRISPRi-mediated gene downregulation, and omics analysis to improve shinorine production, demonstrating the potential of Pseudomonas putida KT2440 as platform for shinorine production.
PubMed: 38839037
DOI: 10.1016/j.ymben.2024.06.001 -
The Journal of Biological Chemistry Feb 2024Bacterial lifestyles depend on conditions encountered during colonization. The transition between planktonic and biofilm growth is dependent on the intracellular second...
Bacterial lifestyles depend on conditions encountered during colonization. The transition between planktonic and biofilm growth is dependent on the intracellular second messenger c-di-GMP. High c-di-GMP levels driven by diguanylate cyclases (DGCs) activity favor biofilm formation, while low levels were maintained by phosphodiesterases (PDE) encourage planktonic lifestyle. The activity of these enzymes can be modulated by stimuli-sensing domains such as Per-ARNT-Sim (PAS). In Pseudomonas aeruginosa, more than 40 PDE/DGC are involved in c-di-GMP homeostasis, including 16 dual proteins possessing both canonical DGC and PDE motifs, that is, GGDEF and EAL, respectively. It was reported that deletion of the EAL/GGDEF dual enzyme PA0285, one of five c-di-GMP-related enzymes conserved across all Pseudomonas species, impacts biofilms. PA0285 is anchored in the membrane and carries two PAS domains. Here, we confirm that its role is conserved in various P. aeruginosa strains and in Pseudomonas putida. Deletion of PA0285 impacts the early stage of colonization, and RNA-seq analysis suggests that expression of cupA fimbrial genes is involved. We demonstrate that the C-terminal portion of PA0285 encompassing the GGDEF and EAL domains binds GTP and c-di-GMP, respectively, but only exhibits PDE activity in vitro. However, both GGDEF and EAL domains are important for PA0285 PDE activity in vivo. Complementation of the PA0285 mutant strain with a copy of the gene encoding the C-terminal GGDEF/EAL portion in trans was not as effective as complementation with the full-length gene. This suggests the N-terminal transmembrane and PAS domains influence the PDE activity in vivo, through modulating the protein conformation.
Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Pseudomonas
PubMed: 38237678
DOI: 10.1016/j.jbc.2024.105659 -
Microbiology Spectrum Dec 2023Gram-negative bacteria from the group are survivors in various environmental niches. For example, the bacteria secrete siderophores to capture ferric ions under...
Gram-negative bacteria from the group are survivors in various environmental niches. For example, the bacteria secrete siderophores to capture ferric ions under deficiency conditions. Tripartite efflux systems are involved in the secretion of siderophores, which are also important for antibiotic resistance. For one of these efflux systems, the resistance-nodulation-cell division transporter ParXY from the model organism KT2440, we show that it influences the secretion of the siderophore pyoverdine in addition to its already known involvement in antibiotic resistance. Phenotypically, its role in pyoverdine secretion is only apparent when other pyoverdine secretion systems are inactive. The results confirm that the different tripartite efflux systems have overlapping substrate specificities and can at least partially functionally substitute for each other, especially in important physiological activities such as supplying the cell with iron ions. This fact must be taken into account when developing specific inhibitors for tripartite efflux systems.
Topics: Siderophores; Pseudomonas putida; Iron; Biological Transport; Ions
PubMed: 37800935
DOI: 10.1128/spectrum.02300-23