-
MicrobiologyOpen Aug 2023Gene inactivation studies are critical in pathogenic bacteria, where insights into species biology can guide the development of vaccines and treatments. Allelic exchange...
Transforming the untransformable with knockout minicircles: High-efficiency transformation and vector-free allelic exchange knockout in the fish pathogen Photobacterium damselae.
Gene inactivation studies are critical in pathogenic bacteria, where insights into species biology can guide the development of vaccines and treatments. Allelic exchange via homologous recombination is a generic method of targeted gene editing in bacteria. However, generally applicable protocols are lacking, and suboptimal approaches are often used for nonstandard but epidemiologically important species. Photobacterium damselae subsp. piscicida (Pdp) is a primary pathogen of fish in aquaculture and has been considered hard to transform since the mid-1990s. Consequently, conjugative transfer of RK2/RP4 suicide vectors from Escherichia coli S17-1/SM10 donor strains, a system prone to off-target mutagenesis, was used to deliver the allelic exchange DNA in previous studies. Here we have achieved efficient electrotransformation in Pdp using a salt-free highly concentrated sucrose solution, which performs as a hypertonic wash buffer, cryoprotectant, and electroporation buffer. High-efficiency transformation has enabled vector-free mutagenesis for which we have employed circular minimalistic constructs (knockout minicircles) containing only allelic exchange essentials that were generated by Gibson assembly. Preparation of competent cells using sucrose and electroporation/integration of minicircles had virtually no detectable off-target promutagenic effect. In contrast, a downstream sacB selection apparently induced several large deletions via mobilization of transposable elements. Electroporation of minicircles into sucrose-treated cells is a versatile broadly applicable approach that may facilitate allelic exchange in a wide range of microbial species. The method permitted inactivation of a primary virulence factor unique to Pdp, apoptogenic toxin AIP56, demonstrating the efficacy of minicircles for difficult KO targets located on the high copy number of small plasmids.
Topics: Animals; Photobacterium; Electroporation; Fishes
PubMed: 37642481
DOI: 10.1002/mbo3.1374 -
Chemosphere Mar 2024Zeolitic imidazolate framework (ZIF) is of wide interest in biomedical applications due to its extraordinary properties such as high storage capacity, functionality and...
Zeolitic imidazolate framework (ZIF) is of wide interest in biomedical applications due to its extraordinary properties such as high storage capacity, functionality and favorable biocompatibility. However, more comprehensive safety assessments are still essential before ZIF is broadly used in biomedicine. Using the characteristic that aldehyde groups on the surface of ZIF-90 can be modified with other functional groups, a series of ZIF-90s modified with different functional groups (oxime group, carboxyl group, amino group and sulfhydryl group) were synthesized to investigate the effect of functionalization on the toxicity of ZIF-90. ZIF-90 series showed concentration-dependent toxic effects on Photobacterium phosphoreum T3 and the functionalized ZIF-90s are more toxic than pristine ZIF-90, with the ZIF-90 modified with amino group (ZIF-90-NH) showing the strongest toxicity (IC = 23.06 mg/L). Based on the results of the cellular assay and stability exploration, we concluded that corresponding imidazole-ligand release and the property of positively charged are responsible for the elevated toxicity of ZIF-90-NH. Cell membrane damage, oxidative damage and luminescence damage are the main contributors to the toxic effects of ZIF-90 series. This study explored the effect of surface functionalization on the toxicity of ZIF and proposed mechanistic clues for the safety application of ZIF.
Topics: Zeolites; Metal-Organic Frameworks; Nanoparticles; Photobacterium
PubMed: 38215832
DOI: 10.1016/j.chemosphere.2024.141188 -
PeerJ 2024Nanoplastics (NPs) are an abundant, long-lasting, and widespread type of environmental pollution that is of increasing concern because of the serious threats they might...
Nanoplastics (NPs) are an abundant, long-lasting, and widespread type of environmental pollution that is of increasing concern because of the serious threats they might pose to ecosystems and species. Identifying the ecological effects of plastic pollution requires understanding the effects of NPs on aquatic organisms. Here, we used the Pacific white shrimp () as a model species to investigate whether ingestion of polystyrene NPs affects gut microbes and leads to metabolic changes in . The abundance of Proteobacteria increased and that of Bacteroidota decreased after NPs treatment. Specifically, spp., spp., spp., and spp. increased in abundance, whereas spp. and spp. decreased. Histological observations showed that exposed to NP displayed a significantly lower intestinal fold height and damaged intestinal structures compared with the control group. Exposure to NPs also stimulated alkaline phosphatase, lysozyme, and acid phosphatase activity, resulting in an immune response in . In addition, the content of triglycerides, total cholesterol, and glucose were significantly altered after NP exposure. These results provided significant ecotoxicological data that can be used to better understand the biological fate and effects of NPs in .
Topics: Animals; Gastrointestinal Microbiome; Ecosystem; Microplastics; Alkaline Phosphatase; Bacteroidetes; Penaeidae
PubMed: 38188162
DOI: 10.7717/peerj.16743 -
Journal of Hazardous Materials Jan 2024In chemical membrane cleaning, the challenge is to efficiently remove irreversible fouling while minimizing the impact on membrane materials. Particularly, traditional...
In chemical membrane cleaning, the challenge is to efficiently remove irreversible fouling while minimizing the impact on membrane materials. Particularly, traditional hypochlorite cleaning will further lead to the generation of toxic halogenated by-products. To address these issues, a combined system composed of peroxymonosulfate and chloride (PMS/Cl) was applied to clean irreversible-humic-acid-fouled polyethersulfone (PES) membranes. After fouled membranes were soaked for 1 h in a PMS/Cl solution (10 mM/15 mM) at 25 °C under neutral conditions, 94% flux recovery and 96% resistance removal were realized. Surface properties of virgin and cleaned membranes were very similar, confirming the effectiveness of the PMS/Cl solution in removing irreversible foulants. The stability of membrane separation performance during multiple fouling and cleaning cycles further confirmed the minimal impact on membrane materials. Rapid diminution of the peaks centered in the region of fulvic-like and humic-like components, monitored under 3D-fluorescence for the cleaning solution, was attributed to PMS-catalyzed chlorination, thereby revealing the primary foulant detachment mechanism. Crucially, the approach exhibited lower toxicity than hypochlorite, as evidenced by reduced halogenated by-products and lower acute toxicity to Photobacterium phosphoreum T3. Overall, this novel cleaning system is promising for the efficient and environmentally friendly removal of irreversible organic foulants in practical water-treatment.
PubMed: 37879274
DOI: 10.1016/j.jhazmat.2023.132827 -
Microorganisms Nov 2023The upsurge in havoc being wreaked by antibiotic-resistant bacteria has led to an urgent need for efficacious alternatives to antibiotics. This study assessed the...
The upsurge in havoc being wreaked by antibiotic-resistant bacteria has led to an urgent need for efficacious alternatives to antibiotics. This study assessed the antibacterial efficacy of two isobutyl cyanoacrylate nanoparticles (iBCA-NPs), D6O and NP30, against major bacterial pathogens of fish. In vivo tests on rainbow trout were preceded by in vitro tests of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). NP30 exhibited higher efficacy than D60, but both iBCA-NPs demonstrated dose-dependent and species-specific in vitro antibacterial properties against the bacterial isolates. Generally, Gram-negative bacteria were more resistant to the iBCA-NPs. , , and were particularly sensitive to both iBCA-NPs. Administered to rainbow trout at 3571.4 mg (iBCA-NP)/kg feed, the iBCA-NPs produced a relative gain rate and survival rates comparable to the control ( > 0.05). The condition factor and the hepatosomatic and viscerosomatic indices of fish were indifferentiable ( > 0.05) between the iBCA-NP groups and the control. The iBCA-NPs caused no alteration in stress, oxidative stress (superoxide dismutase, SOD), plasma complement titer, or lysozyme activity. This study presents the first report of antibacterial activity of iBCA-NPs against Gram-negative bacteria. The results of this study suggest that D60 and NP30 may contribute to reducing the amounts of antibiotics and chemotherapeutic agents used in aquaculture.
PubMed: 38138020
DOI: 10.3390/microorganisms11122877 -
Food Microbiology May 2024We aimed to evaluate the bacterial growth and diversity in vacuum-packed beef bags stored at different temperatures and to monitor blown-pack spoilage. We used...
We aimed to evaluate the bacterial growth and diversity in vacuum-packed beef bags stored at different temperatures and to monitor blown-pack spoilage. We used culture-based methods and high-throughput sequencing to study the development of the main bacterial groups naturally present in beef stored at 4 and 15 °C for 28 days. The growth of sulfite-reducing clostridium (SRC) was impaired in beef bags stored at 4 °C; significant differences among SRC counts were observed in beef bags stored at 4 and 15 °C on days 14, 21, and 28 (P = 0.001). Blown pack was observed in most beef bags stored at 15 °C, from day 14 to day 28, but not in beef bags stored at 4 °C. A storage temperature of 4 °C was able to maintain a stable bacterial microbiota (most prevalent: Photobacterium, Hafnia-Obesumbacterium, and Lactococcus). Remarkable changes in microbial abundance occurred at 15 °C from day 14 to day 28, with a predominance of strict anaerobes (Bacteroides) and the presence of Clostridium spp. The relative frequencies of strict anaerobes and Clostridium were statistically higher in the beef bags stored at 15 °C (P < 0.001 and P = 0.004, respectively). The temperature influenced the microbial counts and relative abundance of spoilage bacteria, leading to blown pack spoilage.
Topics: Animals; Cattle; Food Packaging; Meat; Temperature; Vacuum; Bacteria; Clostridium; Microbiota; Food Microbiology
PubMed: 38225050
DOI: 10.1016/j.fm.2023.104448 -
Frontiers in Immunology 2024Modern fish farming faces challenges in sourcing feed ingredients, most related with their prices, 21 availability, and specifically for plant protein sources,...
INTRODUCTION
Modern fish farming faces challenges in sourcing feed ingredients, most related with their prices, 21 availability, and specifically for plant protein sources, competition for the limited cultivation space for 22 vegetable crops. In that sense, halophytes have the added value of being rich in valuable bioactive compounds and salt tolerant. This study assessed the inclusion of non-food fractions of in European seabass diets.
METHODS
Different levels (2.5%, 5%, and 10%) were incorporated into seabass diets, replacing wheat meal (diets ST2.5, ST5, and ST10) or without inclusion (CTRL). Experimental diets were administered to seabass juveniles (8.62 ± 0.63 g) for 34 and 62 days and subsequent inflammatory responses to a heat-inactivated subsp. () were evaluated in a time-course manner (4, 24, 48, and 72 h after the challenge). At each sampling point, seabass haematological profile, plasma immune parameters, and head-kidney immune-related gene expression were evaluated.
RESULTS
After both feeding periods, most parameters remained unaltered by inclusion; nonetheless, seabass fed ST10 showed an upregulation of macrophage colony-stimulating factor 1 receptor 1 ( and cluster of differentiation 8 ( compared with those fed CTRL after 62 days of feeding. Regarding the inflammatory response, seabass fed ST10 showed lower plasma lysozyme levels than their counterparts fed ST2.5 and ST5 at 24 h following injection, while 4 h after the inflammatory stimulus, seabass fed ST10 presented higher numbers of peritoneal leucocytes than fish fed CTRL. Moreover, at 4 h, fish fed ST2.5, ST5, and ST10 showed a higher expression of interleukin 1β (), while fish fed ST5 showed higher levels of ornithine decarboxylase ( than those fed CTRL. An upregulation of macrophage colony-stimulating factor 1 receptor 1 () and glutathione peroxidase () was also observed at 72 h in fish fed ST10 or ST5 and ST10 compared with CTRL, respectively.
DISCUSSION
In conclusion, incorporating up to 10% of the non-food fraction in feed did not compromise seabass growth or immune status after 62 days, aligning with circular economy principles. However, inclusion improved the leucocyte response and upregulated key immune-related genes in seabass challenged with an inactivated pathogen.
Topics: Animals; Bass; Interleukin-1 Receptor-Like 1 Protein; Macrophage Colony-Stimulating Factor; Diet; Photobacterium
PubMed: 38500885
DOI: 10.3389/fimmu.2024.1342144 -
Food Microbiology Dec 2023This study investigated the combined effect of Ultraviolet (UV) light-emitting diode (LED) technology treatment with refrigerated storage of chicken breast meat over 7...
The efficiency of UV light-emitting diodes (UV-LED) in decontaminating Campylobacter and Salmonella and natural microbiota in chicken breast, compared to a UV pilot-plant scale device.
This study investigated the combined effect of Ultraviolet (UV) light-emitting diode (LED) technology treatment with refrigerated storage of chicken breast meat over 7 days on Campylobacter jejuni, Salmonella enterica serovar Typhimurium, total viable counts (TVC) and total Enterobacteriaceae counts (TEC). An optimised UV-LED treatment at 280 nm for 6 min decreased inoculated S. Typhimurium and C. jejuni populations by 0.6-0.64 log CFU/g, and TVC and TEC population by 1-1.2 log CFU/g in chicken samples. During a 7-day storage at 4 °C, a 0.73 log reduction in C. jejuni was achieved compared with non-treated samples. Moreover, the UV-LED effectiveness to reduce TVC and TEC during refrigerated storage was compared with a conventional UV lamp and a similar efficiency was observed. The impact of UV-LED and UV lamp devices on the microbial community composition of chicken meat during storage was further examined using 16 S rRNA gene amplicon sequencing. Although similar bacterial reductions were observed for both technologies, the microbial communities were impacted differently. Treatment with the UV conventional lamp increased the proportion of Brochothrix spp. In meat samples, whilst Photobacterium spp. Levels were reduced.
Topics: Animals; Campylobacter; Chickens; Ultraviolet Rays; Enterobacteriaceae; Salmonella typhimurium; Microbiota
PubMed: 37689419
DOI: 10.1016/j.fm.2023.104365 -
Water Research Oct 2023The performance of individual reverse osmosis (RO) systems varies significantly with different contaminants of emerging concern (CECs). As such, log reduction values...
The performance of individual reverse osmosis (RO) systems varies significantly with different contaminants of emerging concern (CECs). As such, log reduction values (LRVs) of the concentration of these chemicals cannot be arbitrarily credited in water treatment and water recycling. This study looks to present an approach to the management of chemical risks by providing a systematic validation of RO barrier performance with respect to LRV credits for various classes of CECs. In this work, a one-off sampling campaign across five treatment barriers (strainer filtration, ultrafiltration, RO, ion exchange, chlorination) of a full-scale water recycling plant was conducted, followed by a systematic sampling campaign for a period of six weeks across just the RO barrier. The CECs screening methodology used GC-MS for quantification of 948 trace organic chemicals along with specific 44 per- and polyfluoroalkyl substances (PFAS) screening using LC-MS/MS to demonstrate the removal credits of the RO barrier to a wide spectrum of CECs. The work was used to validate an LRV barrier credit framework so as to predict the performance of a polyamide RO membrane for removal of a range of chemical classes, under typical operational conditions. Conductivity was validated as an efficient surrogate for membrane integrity and RO performance, along with specified operational conditions associated with permeate flux and recovery rate. A bioassay method (photobacterium test) showed good potential to be used as a quick measure to indicate the general toxicity of a sample caused by chemical contamination, because of its high detection sensitivity and time and cost efficiency.
Topics: Chromatography, Liquid; Osmosis; Membranes, Artificial; Tandem Mass Spectrometry; Filtration; Water Purification
PubMed: 37567126
DOI: 10.1016/j.watres.2023.120427 -
Vavilovskii Zhurnal Genetiki I Selektsii Dec 2023At the beginning of the paper, the level of necessary phenomenology of complex models is discussed. When working with complex systems, which of course include living...
At the beginning of the paper, the level of necessary phenomenology of complex models is discussed. When working with complex systems, which of course include living organisms and ecological systems, it is necessary to use a phenomenological description. An illustration of the phenomenological approach is given, which captures the most significant general principles or patterns of interactions; the specific values of the parameters cannot be calculated from the first principles, but are determined empirically. An appropriate interpretation is also chosen empirically and pragmatically. However, in order to simulate a wider range of situations, it becomes necessary to lower the level of phenomenology, switch to a more detailed description of the system, introducing interaction between selected elements of the system. The requirements for a system model combining ecological, metabolic and genetic levels of cell culture description are formulated. A mathematical model of quorum sensing dynamics during the growth of batch culture of luminescent bacteria at different concentrations of the nutrient substrate has been developed. The model contains four blocks describing ecological, energy, quorum and luminescent aspects of bacterial culture growth. The model demonstrated good agreement with the experimental data obtained. When analyzing the model, three oddities in the behavior of the culture were noted, which presumably can change the idea of some processes taking place during the development of a culture of luminescent bacteria. The results obtained suggest the presence of some additional control system for the luminescent reaction via the synthesis pathways of FMN · Н2 or aliphatic aldehyde. In this case, the generalized description of the contribution of energy metabolism to luminescence only through ATP is too strong a simplification. As a result of comparing the model dynamics with the experiment, a discrepancy arose between the concentration of the substrate (peptone) measured in the experiment and its effective influence on the bacterial population growth. This discrepancy seems to indicate peptone is not the leading substrate, and growth is limited by nutrients contained in the yeast extract, the concentration of which did not change in these experiments. The discrepancies noted between the expectations and the results of experimental data processing, together with the assumptions about the causes of these discrepancies, set the direction for further experimental and theoretical studies of quorum sensing mechanisms in a culture of luminescent bacteria.
PubMed: 38500739
DOI: 10.18699/VJGB-23-100