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Applied and Environmental Microbiology Aug 2023Several microbial taxa have been associated with food processing facilities, and they might resist by attaching on tools and equipment even after sanitation procedures,...
Several microbial taxa have been associated with food processing facilities, and they might resist by attaching on tools and equipment even after sanitation procedures, producing biofilms that adhere to the surfaces and might embed other microorganisms, including spoilers and pathogens. There is increasing evidence that these communities can be transferred to the final product. To explore the microbial contamination routes in a facility producing ice creams, we collected foods and environmental swabs from industrial surfaces of equipment and tools and performed taxonomic and functional analyses of the microbial DNA extracted from the environmental samples. Our results suggest that complex communities dominated by psychrotrophic bacteria (e.g., Pseudomonas and Acinetobacter spp.) inhabit the food processing environment, and we demonstrate that these communities might be transferred from the surfaces to the products. Functional analysis performed on environmental samples highlighted the presence of several genes linked to antimicrobial resistance and adherence on abiotic surfaces; such genes were more abundant on food contact (FC) than on other surfaces. Metagenome-assembled genomes (MAGs) of Pseudomonas stutzeri showed genes linked with biofilm formation and motility, which are surely linked to colonizing capabilities in the processing lines. The study highlights clear potential advantages of applying microbiome mapping in the food industry for source tracking of microbial contamination and for planning appropriate sanitization strategies. Several microbial species might permanently establish in food processing facilities, thus contributing to food loss. In fact, food contact surfaces might transfer microorganisms to intermediates and products, potentially representing a hazard to human health. In this work, we provide evidence of the existence of complex microbial communities overcoming sanitation in an ice cream-producing facility. These communities harbored several genes that could potentially lead to attachment to surfaces and antimicrobial resistance. Also, prediction of routes of contamination showed that several potential spoilage taxa might end up in the final product. Importantly, in this work, we show that mapping the environmental microbiome is a high-resolution technique that might help food business operators ensure food quality and safety through detection of potentially hazardous microorganisms.
Topics: Humans; Ice Cream; Virulence; Bacteria; Food Handling; Biofilms; Anti-Infective Agents; Food Microbiology
PubMed: 37432121
DOI: 10.1128/aem.00765-23 -
Scientific Reports Feb 2020Biological ammonium removal via heterotrophic nitrification/aerobic denitrification (HN/AD) presents several advantages in relation to conventional removal processes,...
Physicochemical characterization of Pseudomonas stutzeri UFV5 and analysis of its transcriptome under heterotrophic nitrification/aerobic denitrification pathway induction condition.
Biological ammonium removal via heterotrophic nitrification/aerobic denitrification (HN/AD) presents several advantages in relation to conventional removal processes, but little is known about the microorganisms and metabolic pathways involved in this process. In this study, Pseudomonas stutzeri UFV5 was isolated from an activated sludge sample from oil wastewater treatment station and its ammonium removal via HN/AD was investigated by physicochemical and molecular approaches to better understand this process and optimize the biological ammonium removal in wastewater treatment plants. Results showed that P. stutzeri UFV5 removed all the ammonium in 48-72 hours using pyruvate, acetate, citrate or sodium succinate as carbon sources, C/N ratios 6, 8, 10 and 12, 3-6% salinities, pH 7-9 and temperatures of 20-40 °C. Comparative genomics and PCR revealed that genes encoding the enzymes involved in anaerobic denitrification process are present in P. stutzeri genome, but no gene that encodes enzymes involved in autotrophic nitrification was found. Furthermore, transcriptomics showed that none of the known enzymes of autotrophic nitrification and anaerobic denitrification had their expression differentiated and an upregulation of the biosynthesis machinery and protein translation was observed, besides several genes with unknown function, indicating a non-conventional mechanism involved in HN/AD process.
Topics: Aerobiosis; Ammonium Compounds; Bacterial Proteins; Biodegradation, Environmental; Denitrification; Gene Expression Regulation, Bacterial; Heterotrophic Processes; Nitrification; Pseudomonas stutzeri; Sewage; Transcriptome; Wastewater
PubMed: 32042029
DOI: 10.1038/s41598-020-59279-7 -
Frontiers in Bioengineering and... 2023Acetate is a low-cost feedstock for the production of different bio-chemicals. Electrochemical reduction of CO into acetate and subsequent acetate fermentation is a...
Acetate is a low-cost feedstock for the production of different bio-chemicals. Electrochemical reduction of CO into acetate and subsequent acetate fermentation is a promising method for transforming CO into value-added chemicals. However, the significant inhibitory effect of acetate on microbial growth remains a barrier for acetate-based biorefinery. In this study, the deletion of genes involved in L-leucine degradation was found to be beneficial for the growth of A1501 in acetate. (Δ), in which the hydroxymethylglutaryl-CoA lyase catalyzing -hydroxy--methylglutaryl-CoA into acetyl-CoA and acetoacetate was deleted, grew faster than other mutants and exhibited increased tolerance to acetate. Then, the genes from H16 for poly-3-hydroxybutyrate (PHB) biosynthesis were overexpressed in (∆) and the recombinant strain (∆-) can accumulate 0.11 g L PHB from commercial acetate. Importantly, (∆-) can also use CO-derived acetate to produce PHB and the accumulated PHB accounted for 5.42% (w/w) of dried cell weight of (∆-).
PubMed: 38026858
DOI: 10.3389/fbioe.2023.1297431 -
Frontiers in Microbiology 2018species are frequent inhabitants of freshwater environments and colonizers of water supply networks via bioadhesion and biofilm formation. is the species most commonly...
species are frequent inhabitants of freshwater environments and colonizers of water supply networks via bioadhesion and biofilm formation. is the species most commonly associated with human disease, causing a wide variety of infections with links to its presence in freshwater systems. Though several other species are of ecological and public health importance, little knowledge exists regarding environmental abundances of these species. In the present study, an Illumina-based next-generation sequencing (NGS) approach using -specific primers targeting the 16S rRNA gene was evaluated and applied to a set of freshwater samples from different environments including a cooling tower sampled monthly during 2 years. Our approach showed high specificity and accuracy. NGS read counts revealed a precise quantification of and a good correlation with the absolute number of genome copies in a validated genus-specific qPCR assay, demonstrating the ability of the NGS approach to determine both relative and absolute abundances of species and . The characterization of communities in cooling tower water allowed us to identify 43 phylotypes, with being the most abundant. A shift existed within each year from a community dominated by phylotypes belonging to and phylogenetic groups to a community where was highly abundant. Co-occurrence was observed between and other phylotypes of group as well as the potentially pathogenic species , but not with phylotypes of the group, indicating the need to further investigate the metabolic networks and ecological traits of species. This study demonstrates the potential of deep sequencing as a valuable tool in environmental diagnostics and surveillance of health-related pathogens in freshwater environments.
PubMed: 30186269
DOI: 10.3389/fmicb.2018.01958 -
Microbes and Environments Sep 2016Plant-associated microbes have specific beneficial functions and are considered key drivers for plant health. The bacterial community structure of healthy Anthurium...
Plant-associated microbes have specific beneficial functions and are considered key drivers for plant health. The bacterial community structure of healthy Anthurium andraeanum L. plants was studied by 16S rRNA gene pyrosequencing associated with different plant parts and the rhizosphere. A limited number of bacterial taxa, i.e., Sinorhizobium, Fimbriimonadales, and Gammaproteobacteria HTCC2089 were enriched in the A. andraeanum rhizosphere. Endophytes were more diverse in the roots than in the shoots, whereas all shoot endophytes were found in the roots. Streptomyces, Flavobacterium succinicans, and Asteroleplasma were only found in the roots, Variovorax paradoxus only in the stem, and Fimbriimonas 97%-OTUs only in the spathe, i.e., considered specialists, while Brevibacillus, Lachnospiraceae, Pseudomonas, and Pseudomonas pseudoalcaligenes were generalist and colonized all plant parts. The anaerobic diazotrophic bacteria Lachnospiraceae, Clostridium sp., and Clostridium bifermentans colonized the shoot system. Phylotypes belonging to Pseudomonas were detected in the rhizosphere and in the substrate (an equiproportional mixture of soil, cow manure, and peat), and dominated the endosphere. Pseudomonas included nine 97%-OTUs with different patterns of distribution and phylogenetic affiliations with different species. P. pseudoalcaligenes and P. putida dominated the shoots, but were also found in the roots and rhizosphere. P. fluorescens was present in all plant parts, while P. resinovorans, P. denitrificans, P. aeruginosa, and P. stutzeri were only detected in the substrate and rhizosphere. The composition of plant-associated bacterial communities is generally considered to be suitable as an indicator of plant health.
Topics: Aerobiosis; Anaerobiosis; Araceae; Bacteria; Biota; Cluster Analysis; DNA, Bacterial; DNA, Ribosomal; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Soil Microbiology
PubMed: 27524305
DOI: 10.1264/jsme2.ME16099 -
Journal of Microbiology and... Feb 2017The complexity of the bacterial recombination system is a barrier for the construction of bacterial mutants for the further functional investigation of specific genes....
The complexity of the bacterial recombination system is a barrier for the construction of bacterial mutants for the further functional investigation of specific genes. Several protocols have been developed to inactivate genes from the genus . Those protocols are complicated and time-consuming and mostly do not enable easy construction of multiple knock-ins/outs. The current study describes a single and double crossover-recombination system using an optimized vector-free allele-exchange protocol for gene disruption and gene replacement in a single species of the family Pseudomonadaceae. The protocol is based on self-ligation (circularization) for the DNA cassette which has been obtained by overlapping polymerase chain reaction (Fusion-PCR), and carries an antibiotic resistance cassette flanked by homologous internal regions of the target locus. To establish the reproducibility of the approach, three different chromosomal genes () were knocked-out from the root-associative bacterium A1501. The results showed that the A1501 mutants, which are free of any plasmid backbone, could be obtained via a single or double crossover recombination. In order to optimize this protocol, three key factors that were found to have great effect on the efficiency of the homologous recombination were further investigated. Moreover, the modified protocol does not require further cloning steps, and it enables the construction of multiple gene knock-in/out mutants sequentially. This work provides a simple and rapid mutagenesis strategy for genome editing in , which may also be applicable for other gram-negative bacteria.
Topics: Alleles; Drug Resistance, Bacterial; Gene Editing; Gene Knockout Techniques; Genes, Bacterial; Genetic Engineering; Genetic Vectors; Genome, Bacterial; Mutagenesis; Plasmids; Polymerase Chain Reaction; Pseudomonas stutzeri; Recombination, Genetic
PubMed: 27817190
DOI: 10.4014/jmb.1608.08019 -
Frontiers in Medicine 2023Although pre/pro/postbiotics have become more prevalent in dermatologic and cosmetic fields, the mode of action when topically applied is largely unknown. A multi-omic...
INTRODUCTION
Although pre/pro/postbiotics have become more prevalent in dermatologic and cosmetic fields, the mode of action when topically applied is largely unknown. A multi-omic approach was applied to decipher the impact of the skincare products with pre/postbiotics on skin microbiome and metabolome.
METHODS
Subjects with dry skin applied a body wash and body lotion with or without pre/postbiotics for 6 weeks. Skin hydration was measured at baseline, 3 and 6 weeks. Skin swabs were collected for 16S rRNA gene sequencing, metagenomics and metabolomics analysis.
RESULTS
Skin hydration significantly increased in both groups. The prebiotic group significantly reduced opportunistic pathogens, e.g., and , and increased the commensals, e.g., , . Bacterial sugar degradation pathways were enriched in the prebiotic group, while fatty acid biosynthesis pathways were reduced in control. The changes on skin metabolome profiles by the products were more prominent. The prebiotic group performed greater modulation on many clinically-relevant metabolites compared to control. Correlation analysis showed and positively correlated with skin hydration, and negatively correlated with the metabolites that are positively associated with skin hydration improvement.
CONCLUSION
This holistic study supported a hypothesis that the pre/postbiotics increased skin hydration through the modulation of skin microbiome, metabolic pathways and metabolome.
PubMed: 37534320
DOI: 10.3389/fmed.2023.1165980 -
Journal of Microbiology and... Aug 2015Until now, considerable effort has been made to engineer novel nitrogen-fixing organisms through the transfer of nif genes from various diazotrophs to non-nitrogen...
Until now, considerable effort has been made to engineer novel nitrogen-fixing organisms through the transfer of nif genes from various diazotrophs to non-nitrogen fixers; however, regulatory coupling of the heterologous nif genes with the regulatory system of the new host is still not well understood. In this work, a 49 kb nitrogen fixation island from P. stutzeri A1501 was transferred into E. coli using a novel and efficient transformation strategy, and a series of recombinant nitrogen-fixing E. coli strains were obtained. We found that the nitrogenase activity of the recombinant E. coli strain EN-01, similar to the parent strain P. stutzeri A1501, was dependent on external ammonia concentration, oxygen tension, and temperature. We further found that there existed a regulatory coupling between the E. coli general nitrogen regulatory system and the heterologous P. stutzeri nif island in the recombinant E. coli strain. We also provided evidence that the E. coli general nitrogen regulator GlnG protein was involved in the activation of the nif-specific regulator NifA via a direct interaction with the NifA promoter. To the best of our knowledge, this work plays a groundbreaking role in increasing understanding of the regulatory coupling of the heterologous nitrogen fixation system with the regulatory system of the recipient host. Furthermore, it will shed light on the structure and functional integrity of the nif island and will be useful for the construction of novel and more robust nitrogen-fixing organisms through biosynthetic engineering.
Topics: Ammonia; Escherichia coli; Gene Expression Regulation, Bacterial; Gene Regulatory Networks; Gene Transfer, Horizontal; Genomic Islands; Metabolic Networks and Pathways; Nitrogen Fixation; Oxygen; Pseudomonas stutzeri; Temperature
PubMed: 25824431
DOI: 10.4014/jmb.1502.02027 -
MicrobiologyOpen Apr 2020Heterologous production of extracellular polyhydroxybutyrate (PHB) depolymerases (PhaZs) has been of interest for over 30 years, but implementation is sometimes...
Heterologous production of extracellular polyhydroxybutyrate (PHB) depolymerases (PhaZs) has been of interest for over 30 years, but implementation is sometimes difficult and can limit the scope of research. With the constant development of tools to improve recombinant protein production in Escherichia coli, we propose a method that takes characteristics of PhaZs from different bacterial strains into account. Recombinant His-tagged versions of PhaZs (rPhaZ) from Comamonas testosteroni 31A, Cupriavidus sp. T1, Marinobacter algicola DG893, Pseudomonas stutzeri, and Ralstonia sp. were successfully produced with varying expression, solubility, and purity levels. PhaZs from C. testosteroni and P. stutzeri were more amenable to heterologous expression in all aspects; however, using the E. coli Rosetta-gami B(DE3) expression strain and establishing optimal conditions for expression and purification (variation of IPTG concentration and use of size exclusion columns) helped circumvent low expression and purity for the other PhaZs. Degradation activity of the rPhaZs was compared using a simple PHB plate-based method, adapted to test for various pH and temperatures. rPhaZ from M. algicola presented the highest activity at 15°C, and rPhaZs from Cupriavidus sp. T1 and Ralstonia sp. had the highest activity at pH 5.4. The methods proposed herein can be used to test the production of soluble recombinant PhaZs and to perform preliminary evaluation for applications that require PHB degradation.
Topics: Bacteria; Bioreactors; Carboxylic Ester Hydrolases; Comamonas testosteroni; Cupriavidus; Escherichia coli; Marinobacter; Pseudomonas stutzeri; Ralstonia; Recombinant Proteins
PubMed: 32087608
DOI: 10.1002/mbo3.1001 -
Microorganisms Nov 2021Integrative and conjugative elements (ICEs) are chromosomally integrated self-transmissible mobile genetic elements. Although some ICEs are known to carry genes for the...
A New ICE Subfamily Integrative and Conjugative Element Responsible for Horizontal Transfer of Biphenyl and Salicylic Acid Catabolic Pathway in the PCB-Degrading Strain KF716.
Integrative and conjugative elements (ICEs) are chromosomally integrated self-transmissible mobile genetic elements. Although some ICEs are known to carry genes for the degradation of aromatic compounds, information on their genetic features is limited. We identified a new member of the ICE family carrying biphenyl catabolic genes and salicylic acid catabolic genes from the PCB-degrading strain KF716. The 117-kb ICEKF716 contains common core regions exhibiting homology with those of degradative ICE from B13 and ICE from sp. CIB. A comparison of the gene loci collected from the public database revealed that several putative ICEs from B6-2 JAB1, AN10 and 2A20 had highly conserved core regions with those of ICEKF716, along with the variable region that encodes the catabolic genes for biphenyl, naphthalene, toluene, or phenol. These data indicate that this type of ICE subfamily is ubiquitously distributed within aromatic compound-degrading bacteria. ICEKF716 was transferred from KF716 to PAO1 via a circular extrachromosomal intermediate form. In this study, we describe the structure and genetic features of ICEKF716 compared to other catabolic ICEs.
PubMed: 34946064
DOI: 10.3390/microorganisms9122462