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Journal of Dairy Science Sep 2020The objective of this study was to investigate the effect of adding different levels of a thermoresistant protease produced by a Pseudomonas fluorescens strain to milk...
The objective of this study was to investigate the effect of adding different levels of a thermoresistant protease produced by a Pseudomonas fluorescens strain to milk on the manufacture and quality of Cheddar cheese. Fresh raw milk was collected, standardized, and pasteurized at 72°C for 15 s, and the enzyme was added to give a protease activity of 0.15 or 0.60 U/L (treatments P1 and P4, respectively), while one sample had no enzyme added (control). Milk was stored at 4°C for 48 h and Cheddar cheese was manufactured after 0 and 48 h of storage. Results indicated that the protease was active in milk during 48 h of storage; however, its effect on milk composition was minimal. The protein that was preferentially hydrolyzed by the protease over storage was β-casein, followed by κ-casein. The mean cheese yield and recovery of fat and protein obtained for all cheeses were not affected by protease activity. The protease showed low activity during cheese manufacture, possibly because of unfavorable conditions, including low pH. One of the factors that might have influenced protease activity was the pH of the curd (approximately 6.55 after acidification and 5.35 at milling), which was lower than that at which the enzyme would have optimum activity (pH 7 to 9). Consequently, the composition, pH, patterns of proteolysis, and hardness of all cheeses produced were similar and in accordance with values expected for that type of cheese, independently of the protease activity level. However, slight increases in proteolysis were observed in P4 cheeses and produced using milk stored for 48 h. Both the P1 and P4 cheeses had higher concentrations of free amino acids (FAA) compared with the control, whereas urea-PAGE electrophoretograms indicated a greater breakdown of caseins in the P4 cheese samples, which may be related to possible increases in numbers of proteolytic bacteria in milk during storage. Therefore, the thermoresistant psychrotrophic bacterial protease(s) tested in this study may affect the manufacture or quality of Cheddar cheese during ripening to a relatively limited extent. However, controlling initial levels of proteolytic bacteria in raw milk remains essential, because proteolysis affects the development of flavor and texture in cheese.
Topics: Animals; Caseins; Cheese; Food Quality; Hydrogen-Ion Concentration; Milk; Pasteurization; Peptide Hydrolases; Proteolysis; Pseudomonas fluorescens; Taste
PubMed: 32600766
DOI: 10.3168/jds.2019-18043 -
Journal of Dairy Science Mar 2021Pseudomonas spp. are important spoilage bacteria that negatively affect the quality of refrigerated fluid milk and uncultured cheese by generating unwanted odors,...
Pseudomonas spp. are important spoilage bacteria that negatively affect the quality of refrigerated fluid milk and uncultured cheese by generating unwanted odors, flavors, and pigments. They are frequently found in dairy plant environments and enter dairy products predominantly as postpasteurization contaminants. Current subtyping and characterization methods for dairy-associated Pseudomonas are often labor-intensive and expensive or provide limited and possibly unreliable classification information (e.g., to the species level). Our goal was to identify a single-copy gene that could be analyzed in dairy spoilage-associated Pseudomonas for preliminary species-level identification, subtyping, and phenotype prediction. We tested 7 genes previously targeted in a Pseudomonas fluorescens multilocus sequence typing scheme for their individual suitability in this application using a set of 113 Pseudomonas spp. isolates representing the diversity of typical pasteurized milk contamination. For each of the 7 candidate genes, we determined the success rate of PCR and sequencing for these 113 isolates as well as the level of discrimination for species identification and subtyping that the sequence data provided. Using these metrics, we selected a single gene, isoleucyl tRNA synthetase (ileS), which had the most suitable traits for simple and affordable single-gene Pseudomonas characterization. This was based on the number of isolates successfully sequenced for ileS (113/113), the number of unique allelic types assigned (83, compared with 50 for 16S rDNA), nucleotide and sequence diversity measures (e.g., number of unique SNP and Simpson index), and tests for genetic recombination. The discriminatory ability of ileS sequencing was confirmed by separation of 99 additional dairy Pseudomonas spp. isolates, which were indistinguishable by 16S rDNA sequencing, into 28 different ileS allelic types. Further, we used whole-genome sequencing data to demonstrate the similarities in ileS-based phylogenetic clustering to whole-genome-based clustering for 27 closely related dairy-associated Pseudomonas spp. isolates and for 178 Pseudomonas type strains. We also found that dairy-associated Pseudomonas within an ileS cluster typically shared the same proteolytic and lipolytic activities. Use of ileS sequencing provides a promising strategy for affordable initial characterization of Pseudomonas isolates, which will help the dairy industry identify, characterize, and track Pseudomonas in their facilities and products.
Topics: Animals; Dairying; Food Contamination; Isoleucine-tRNA Ligase; Milk; Phylogeny; Pseudomonas
PubMed: 33455773
DOI: 10.3168/jds.2020-19283 -
Scientific Reports Aug 2023The compound 2,4-diacetylphloroglucinol (DAPG) is a broad-spectrum antibiotic that is primarily produced by Pseudomonas spp. DAPG plays an important role in the...
The compound 2,4-diacetylphloroglucinol (DAPG) is a broad-spectrum antibiotic that is primarily produced by Pseudomonas spp. DAPG plays an important role in the biocontrol disease suppressing activity of Pseudomonas spp. In the current study, we report the discovery of the DAPG biosynthetic cluster in strains of Chromobacterium vaccinii isolated from Brazilian aquatic environments and the distribution of the biosynthetic cluster in the Chromobacterium genus. Phylogenetic analysis of the phlD protein suggests the biosynthetic cluster probably entered the genus of Chromobacterium after a horizontal gene transfer event with a member of the Pseudomonas fluorescens group. We were able to detect trace amounts of DAPG in wild type cultures and confirm the function of the cluster with heterologous expression in Escherichia coli. In addition, we identified and verified the presence of other secondary metabolites in these strains. We also confirmed the ability of C. vaccinii strains to produce bioactive pigment violacein and bioactive cyclic depsipeptide FR900359. Both compounds have been reported to have antimicrobial and insecticidal activities. These compounds suggest strains of C. vaccinii should be further explored for their potential as biocontrol agents.
Topics: Chromobacterium; Phylogeny; Anti-Bacterial Agents; Brazil; Escherichia coli; Pseudomonas
PubMed: 37653049
DOI: 10.1038/s41598-023-41277-0 -
Microbial Genomics Jul 2021is a highly diverse genus that includes species that cause disease in both plants and animals. Recently, pathogenic pseudomonads from the and species complexes have...
is a highly diverse genus that includes species that cause disease in both plants and animals. Recently, pathogenic pseudomonads from the and species complexes have caused significant outbreaks in several agronomically important crops in Turkey, including tomato, citrus, artichoke and melon. We characterized 169 pathogenic strains associated with recent outbreaks in Turkey via multilocus sequence analysis and whole-genome sequencing, then used comparative and evolutionary genomics to characterize putative virulence mechanisms. Most of the isolates are closely related to other plant pathogens distributed among the primary phylogroups of , although there are significant numbers of isolates, which is a species better known as a rhizosphere-inhabiting plant-growth promoter. We found that all 39 citrus blast pathogens cluster in phylogroup 2, although strains isolated from the same host do not cluster monophyletically, with lemon, mandarin orange and sweet orange isolates all being intermixed throughout the phylogroup. In contrast, 20 tomato pith pathogens are found in two independent lineages: one in the secondary phylogroups, and the other from the species complex. These divergent pith necrosis strains lack characteristic virulence factors like the canonical tripartite type III secretion system, large effector repertoires and the ability to synthesize multiple bacterial phytotoxins, suggesting they have alternative molecular mechanisms to cause disease. These findings highlight the complex nature of host specificity among plant pathogenic pseudomonads.
Topics: Crops, Agricultural; Genome, Bacterial; Multilocus Sequence Typing; Plant Diseases; Plants; Pseudomonas fluorescens; Pseudomonas syringae; Turkey; Type III Secretion Systems; Virulence Factors; Whole Genome Sequencing
PubMed: 34227931
DOI: 10.1099/mgen.0.000585 -
Journal of Bacteriology Sep 2023Biofilm formation by the Gram-negative, Gammaproteobacteria relies on the repeats-in-toxin adhesins LapA and MapA in the cytoplasm, secretion of these adhesins through...
Biofilm formation by the Gram-negative, Gammaproteobacteria relies on the repeats-in-toxin adhesins LapA and MapA in the cytoplasm, secretion of these adhesins through their respective type 1 secretion systems, and retention at the cell surface. Published work has shown that retention of the adhesins occurs via a post-translational mechanism involving the cyclic-di-GMP receptor LapD and the protease LapG. However, little is known about the underlying mechanisms that regulate the level of these adhesins. Here, we demonstrate that the master regulator FleQ modulates biofilm formation by both transcriptionally and post-transcriptionally regulating LapA and MapA. We find that a Δ mutant has a biofilm formation defect compared to the wild-type (WT) strain, which is attributed in part to a decrease in LapA and MapA abundance in the cell, despite the Δ mutant having increased levels of and transcripts compared to the WT strain. Through transposon mutagenesis and subsequent genetic analysis, we found that overstimulation of the Gac/Rsm pathway partially rescues biofilm formation in the Δ mutant background. Collectively, these findings provide evidence that FleQ regulates biofilm formation by both transcriptionally regulating the expression of the genes and post-transcriptionally regulating the abundance of LapA and MapA, and that activation of the Gac/Rsm pathway can post-transcriptionally enhance biofilm formation by . IMPORTANCE Biofilm formation is a highly coordinated process that bacteria undergo to colonize a variety of surfaces. For , biofilm formation requires the production and localization of repeats-in-toxin adhesins to the cell surface. To date, little is known about the underlying mechanisms that regulate biofilm formation by . Here, we identify FleQ as a key regulator of biofilm formation that modulates both gene expression and abundance of LapA and MapA through both a transcriptional and post-transcriptional mechanism. We provide further evidence implicating activation of the Gac/Rsm system in FleQ-dependent regulation of biofilm formation. Together, our findings uncover evidence for a dual mechanism of transcriptional and post-transcriptional regulation of the LapA and MapA adhesins.
Topics: Pseudomonas fluorescens; Biofilms; Adhesins, Bacterial; Carrier Proteins; Bacterial Proteins; Gene Expression Regulation, Bacterial; Cyclic GMP
PubMed: 37655913
DOI: 10.1128/jb.00152-23 -
Frontiers in Microbiology 2020Metallothioneins (MTs) are low molecular weight, Cys-rich proteins that sequester both essential and non-essential metal ions. Despite being highly conserved in the...
Metallothioneins (MTs) are low molecular weight, Cys-rich proteins that sequester both essential and non-essential metal ions. Despite being highly conserved in the genus of Gram-negative bacteria, knowledge of their physiological function in this species is scarce. Using the strain Q2-87 as a model organism, we investigated the role of a conserved MT in zinc homeostasis, cadmium detoxification as well as its implications in stress response. We show that MT expression is only induced in the stationary phase and provides a fitness benefit for long-term starvation survival, while it is not required for metal resistance and acquisition, oxidative or nitrosative stress response, biofilm formation or motility.
PubMed: 32793167
DOI: 10.3389/fmicb.2020.01709 -
Anais Da Academia Brasileira de Ciencias 2023Pseudomonas fluorescens is known to have the ability to adhere and produce biofilm. The formation of biofilms is enhanced by cellular motility, particularly when...
Pseudomonas fluorescens is known to have the ability to adhere and produce biofilm. The formation of biofilms is enhanced by cellular motility, particularly when mediated by flagella. Biofilm formed on surfaces such as those used for food production act as points of contamination, releasing pathogenic or deteriorating microorganisms and compromising the quality of products. We assessed two strains of Pseudomonas fluorescens PL5.4 and PL7.1, sampled from raw, chilled, buffalo milk, which was obtained from a dairy farm. Twitching and swarming motility assays were performed, in addition to the biofilm production evaluations at a temperature of 7 °C. Regarding the motility assays, only the PL5.4 strain scored positive for the swarming assay. On microplates, both strains presented themselves as strong biofilm producers at 7 °C. The PL5.4 strain was also able to form biofilm on a stainless steel structure and maintain this structure for up to 72 hours at refrigeration. The Pseudomonas fluorescens PL5.4 isolate was identified on the basis of a 99% sequence identity with Pseudomonas fluorescens A506, a strain used as a biocontrol in agriculture. Biofilm-forming bacteria, when adapted to low temperatures, become a constant source of contamination, damaging the production, quality, safety and shelf-life of products.
Topics: Animals; Pseudomonas fluorescens; Milk; Biofilms; Temperature
PubMed: 37466543
DOI: 10.1590/0001-3765202320220982 -
Biofilm Dec 2023Biofilms are complex microbial communities embedded in extracellular matrix. Pathogens within the biofilm become more resistant to the antibiotics than planktonic...
Biofilms are complex microbial communities embedded in extracellular matrix. Pathogens within the biofilm become more resistant to the antibiotics than planktonic counterparts. Novel strategies are required to encounter biofilms. Exopolysaccharides are one of the major components of biofilm matrix and play a vital role in biofilm architecture. In previous studies, a glycosyl hydrolase, PslG, from was found to be able to inhibit biofilm formation by disintegrating exopolysaccharide in biofilms. Here, we investigate the potential spectrum of PslG homologous protein with anti-biofilm activity. One glycosyl hydrolase from , PslG, exhibits anti-biofilm activities and the key catalytic residues of PslG are conserved with those of PslG. PslG at concentrations as low as 50 nM efficiently inhibits the biofilm formation of and disassemble its preformed biofilm. Furthermore, PslG exhibits anti-biofilm activity on a series of , including and pv. . PslG stays active under various temperatures. Our findings suggest that glycosyl hydrolase PslG has potential to be a broad spectrum inhibitor on biofilm formation of a wide range of .
PubMed: 37928620
DOI: 10.1016/j.bioflm.2023.100155 -
Food Research International (Ottawa,... Feb 2023To better understand the microbial quality and safety of plant-based meat analogues, this study investigated the changes of native microflora present in soy- and...
To better understand the microbial quality and safety of plant-based meat analogues, this study investigated the changes of native microflora present in soy- and pea-based meat analogues (SBM and PBM) and compared them with ground beef (GB). SBM, PBM, and GB were also artificially inoculated with meat spoilage microorganisms, Pseudomonas fluorescens and Brochothrix thermosphacta, and pathogenic microorganisms, Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes; the fitness of these bacteria was evaluated during storage at refrigerated and/or abused temperatures. Results showed that the initial total aerobic plate count (APC), coliform, lactic acid bacteria (LAB), and mold/yeast (M/Y) counts for GB could be as high as 5.44, 2.90, 4.61, and 3.45 log CFU/g, while the highest initial APC, coliform, LAB, and M/Y counts found in SBM were 3.10, 2.00, 2.04, and 1.95 log CFU/g, and were 3.82, 2.51, 3.61, and 1.44 log CFU/g for PBM. The batch-to-batch differences in microbial counts were more significant in GB than in SBM and PBM. Despite the different initial concentrations, there was no difference among APC and LAB counts between the three meat types by the end of the 10-day 4 °C storage period, all approaching ca. 7.00 log CFU/g. Artificially-inoculated B. thermosphacta increased by 0.76, 1.58, and 0.96 log CFU/g in GB, PBM, and SBM respectively by the end of the refrigeration storage; P. fluorescens increased by 4.92, 3.00, and 0.40 log CFU/g in GB, PBM, and SBM respectively. Under refrigerated storage conditions, pathogenic bacteria did not change in GB and SBM. L. monocytogenes increased by 0.74 log in PBM during the 7-day storage at 4 °C. All three pathogens grew at abused storage temperatures, regardless of the meat type. Results indicated that plant-based meat could support the survival and even growth of spoilage and pathogenic microorganisms. Preventive controls are needed for ensuring the microbial quality and safety of plant-based meat analogues.
Topics: Animals; Cattle; Meat Products; Food Microbiology; Meat; Salmonella; Pseudomonas fluorescens
PubMed: 36737989
DOI: 10.1016/j.foodres.2022.112408 -
Frontiers in Microbiology 2022is considered a foodborne pathogen of serious concern capable of forming multispecies biofilms with other bacterial species, such as spp., adhered onto stainless steel...
is considered a foodborne pathogen of serious concern capable of forming multispecies biofilms with other bacterial species, such as spp., adhered onto stainless steel (SS) surfaces. In an attempt to link the biofilms' morphology and resistance to biocides, dual-species biofilms of , in co-culture with either , , or , were assayed to ascertain their morphological characteristics and resistance toward benzalkonium chloride (BAC) and neutral electrolyzed water (NEW). Epifluorescence microscopy analysis revealed that each dual-species biofilm was distributed differently over the SS surface and that these differences were attributable to the presence of spp. Confocal laser scanning microscopy (CLSM) assays demonstrated that despite these differences in distribution, all biofilms had similar maximum thicknesses. Along with this, colocalization analyses showed a strong trend of to share location within the biofilm with all assayed whilst the latter distributed throughout the surface independently of the presence of , a fact that was especially evident in those biofilms in which cell clusters were present. Finally, a modified Gompertz equation was used to fit biofilms' BAC and NEW dose-response data. Outcomes demonstrated that was less susceptible to BAC when co-cultured with or , whereas susceptibility to NEW was reduced in all three dual-species biofilms, which can be attributable to both the mechanism of action of the biocide and the architectural features of each biofilm. Therefore, the results herein provided can be used to optimize already existing and develop novel target-specific sanitation treatments based on the mechanism of action of the biocide and the biofilms' species composition and structure.
PubMed: 35756028
DOI: 10.3389/fmicb.2022.917964