Culturable Cells Research Articles

Clinical Trial of Autologous Cultivated Limbal Epithelial Cell Sheet Transplantation for Patients with Limbal Stem Cell Deficiency

To confirm the efficacy and safety of Good Manufacturing Practice (GMP)-compliant autologous cultivated limbal epithelial cell sheets in government-controlled clinical trials that adhered to Good Clinical Practice stipulations for patients with unilateral limbal stem cell deficiency (LSCD). A prospective, multicenter, open-label, uncontrolled, single-arm clinical trial. Ten consecutive eyes of 10 patients with unilateral LSCD were followed for 2 years after surgery. Preoperative LSCD stage was IIB in 4 eyes and III in 6 eyes. A limbal tissue biopsy was obtained from the healthy eye, after which limbal stem cells were dissociated and cultivated on temperature-responsive culture surfaces. All cell sheets were fabricated in a GMP-grade facility under established standard operating procedures. Cell sheets were evaluated using defined shipment criteria before transplantation, and only those that met the criteria were used. The cell sheet was transplanted onto each of the patients' diseased eye after removing the conjunctival scar tissue that covered the corneal surface. The severity of LSCD was determined according to a staging method agreed on by global consensus, with eyes evaluated as being in stages IA-C representing successful corneal epithelial reconstruction. Diagnosis and staging of LSCD were determined by the trial's Eligibility Judgment Committee and Effect Assessment Committee using slit-lamp photographs including fluorescein staining. Both committees comprised 2 or 3 third-party cornea specialists, who were provided with information anonymously and randomly. Corneal epithelial reconstruction rate was the primary end point. Corneal epithelial reconstruction was successful in 6 of 10 eyes (60%) 1 year postoperatively and was significantly higher than the 15% clinically significant efficacy rate achieved by allogeneic limbal transplantation. The reconstruction rate was 70% of eyes 2 years postoperatively. Additionally, improvements in visual acuity were noted in 50% and 60% of eyes at 1 and 2 years, respectively. No clinically significant transplantation-related adverse events were observed. The efficacy and safety of cultivated limbal epithelial cell sheet transplantation were thus confirmed, and the cell sheet, named "Nepic," is now approved as a cellular and tissue-based product in Japan. Proprietary or commercial disclosure may be found after the references. Proprietary or commercial disclosure may be found after the references.

Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR.

Legionella pneumophila is a waterborne pathogen and, as the causative agent of Legionnaires' disease, a significant public health concern. Exposure to environmental stresses, and disinfection treatments, promotes the formation of resistant and potentially infectious viable but non-culturable (VBNC) Legionella. The management of engineered water systems to prevent Legionnaires' disease is hindered by the presence of VBNC Legionella that cannot be detected using the standard culture (ISO11731:2017-05) and quantitative polymerase reaction (ISO/TS12869:2019) methods. This study describes a novel method to quantify VBNC Legionella from environmental water samples using a "viability based flow cytometry-cell sorting and qPCR" (VFC + qPCR) assay. This protocol was then validated by quantifying the VBNC Legionella genomic load from hospital water samples. The VBNC cells were unable to be cultured on Buffered Charcoal Yeast Extract (BCYE) agar; however, their viability was confirmed through their ATP activity and ability to infect amoeba hosts. Subsequently, an assessment of the ISO11731:2017-05 pre-treatment procedure demonstrated that acid or heat treatment cause underestimation of alive Legionella population. Our results showed that these pre-treatment procedures induce culturable cells to enter a VBNC state. This may explain the observed insensitivity and lack of reproducibility often observed with the Legionella culture method. This study represents the first time that flow cytometry-cell sorting in conjunction with a qPCR assay has been used as a rapid and direct method to quantify VBNC Legionella from environmental sources. This will significantly improve future research evaluating Legionella risk management approaches for the control of Legionnaires' disease.

Understanding Ocular Surface Inflammation in Tears Before and After Autologous Cultivated Limbal Epithelial Stem Cell Transplantation.

We aimed to determine the expression of inflammatory cytokines in the tears of patients with unilateral total limbal stem cell deficiency (TLSCD) caused by chemical burns before and after autologous cultivated limbal epithelial stem cell transplantation (CLET). Tear samples were collected from both eyes of 23 patients with unilateral TLSCD and 11 healthy controls, at fixed timepoints before and after CLET. Dissolved molecules were extracted from Schirmer's strips using a standardised method and analysed on an array plate of ten inflammatory cytokines (V-Plex Proinflammatory Panel 1 Human Kit, MSD). IL1β expression was significantly elevated in the TLSCD eye compared with the unaffected eye at baseline (p < 0.0001) but decreased to normal 3months post-CLET (p = 0.22). IL6 and IL8 were unaffected at baseline but significantly elevated in the TLSCD eyes at 1month post-CLET (p = 0.001 and p < 0.0001, respectively). IL6 returned to normal at 3months and IL8 at 6months post-CLET. There was a significant renewed increase in IL1β, IL6 and IL8 expression at 12months post-CLET (p < 0.0001, p = 0.0001 and p = 0.0003, respectively). IFNγ, IL10 and IL12p70 expression were significantly reduced in both eyes of patients with unilateral TLSCD at all timepoints. IL1β is a specific marker of inflammation in TLSCD eyes that could be therapeutically targeted pre-CLET to improve stem cell engraftment. At 12months post-CLET the spike in levels of IL1β, IL6 and IL8 coincides with cessation of topical steroids, suggesting ongoing subclinical inflammation. We therefore recommend not discontinuing topical steroid treatment in cases where penetrating keratoplasty is indicated; however, further investigation is needed to ascertain this. European Union Drug Regulating Authorities Clinical Trials Database (EuDRACT 2011-000608-16); ISRCTN (International Standard Randomised Controlled Trial Number (isrctn51772481).

Simultaneous dairy wastewater treatment and bioelectricity production in a new microbial fuel cell using photosynthetic Synechococcus.

Photosynthetic microbial fuel cell (PMFC) is a novel technology, which employs organic pollutants and organisms to produce electrons and biomass and capture CO2 by bio-reactions. In this study, a new PMFC was developed based on Synechococcus sp. as a biocathode, and dairy wastewater was used in the anode chamber. Different experiments including batch feed mode, semi-continuous feed mode, Synechococcus feedstock to the anode chamber, Synechococcus-Chlorella mixed system, the feedstock of treated wastewater to the cathode chamber, and use of extra nutrients in the anodic chamber were performed to investigate the behavior of the PMFC system. The results indicated that the PMFC with a semi-continuous feed mode is more effective than a batch mode for electricity generation and pollutant removal. Herein, maximum power density, chemical oxygen demand removal, and Coulombic efficiency were 6.95 mW/m2 (450 Ω internal resistance), 62.94, and 43.16%, respectively, through mixing Synechococcus sp. and Chlorella algae in the batch-fed mode. The maximum nitrate and orthophosphate removal rates were 98.83 and 68.5%, respectively, wherein treated wastewater in the anode was added to the cathode. No significant difference in Synechococcus growth rate was found between the cathodic chamber of PMFC and the control cultivation cell. The heating value of the biocathode biomass at maximum Synechococcus growth rate (adding glucose into the anode chamber) was 0.2235MJ/Kg, indicating the cell's high ability for carbon dioxide recovery. This study investigated not only simultaneous bioelectricity production and dairy wastewater in a new PMFC using Synechococcus sp. but also studied several operational parameters and presented useful information about their effect on PMFC performance.

Survival, growth, and biogenic amine production of Enterococcus faecium FC12 in response to extracts and essential oils of Rubus fruticosus and Juniperus oxycedrus.

Enterococci are lactic acid bacteria (LAB) usually found as food contaminants in fermented products such as cheeses and fermented sausages. Due to their antibiotic resistance, the presence of virulence factors, and the ability to produce biogenic amines (BAs), the determination of these bacteria is crucial to assure food quality and safety. BAs production and consequent accumulation in foods can cause toxicological effects on human health. Plant phenolic compounds are promising alternatives to chemical preservatives and reflect consumers' demand for "green" solutions. In this study, the antimicrobial effect of blackberry (Rubus fruticosus) leaves and prickly juniper (Juniperus oxycedrus) needles, both as phenolic extracts (PE) and essential oils (EO), were evaluated against Enterococcus faecium FC12, a known tyramine-producing strain. The growth kinetics in the presence of sub-lethal concentrations of such plant derivatives were modeled (Gompertz equation) and BA production was monitored over time by HPLC. Moreover, flow cytometry (FCM) was used to study the effects of EOs and PEs on cell viability. The EOs showed a higher antimicrobial effect (especially R. fruticosus added at 0.75 mg/ml), determining an initial decrease of culturable cells followed by a recovery, even if with lower growth rates and final cell loads. Different rates of BA formation were observed, with tyramine concentrations ranging from 120 to 160 mg/l after 96 h of incubation, and 2-phenylethylamine was produced in lower amounts, usually after reaching the peak of tyramine. FCM confirmed the higher efficacy of R. fruticosus EO that induced cell membrane injury in 93% of the total population. However, complete recovery occurred in the following incubation, demonstrating transient damage. Although further research is required to better investigate this recovery and to assess the suitability of this approach in a real food system, the present study showed the potential antimicrobial activity of plant derivatives, especially R. fruticosus EO, against the tyramine-producing E. faecium FC12.

Cell phenotype changes and oxidative stress response in Vibrio spp. induced into viable but non-culturable (VBNC) state

PurposeAquatic bacteria of the genus Vibrio include animal and human pathogens. The occurrence of Vibrio-related diseases has been associated with the current climate change-driven increase of sea surface temperature. Vibrio spp. can enter into the viable but non-culturable (VBNC) state, as a consequence of starvation in seawater at low temperatures. In such physiological state, Vibrio cells are no longer culturable on standard media agar plates but can resuscitate if incubated at 30 °C prior to plating, retaining virulence. Since limited information is available on regards to this topic, in this work, we characterized the phenotypic changes of four Vibrio spp. strains (one laboratory strain and three environmental isolates) in cold seawater microcosms, investigating the relationship between resuscitation and a hydrogen peroxide-induced oxidative stress.MethodsCell phenotypic changes and the effect of hydrogen peroxide and/or catalase addition to the medium were studied on VBNC and resuscitated cells by flow cytometry in microcosm experiments, paralleled by culturability experiments by plating.ResultsThe cells of all the Vibrio strains changed their phenotype upon the induction of the VBNC state resulting in cell dwarfing and decrease in DNA quantity, losing the ability to grow on solid media. These features were partially or totally reverted when the cells were treated for resuscitation. Hydrogen peroxide at concentrations as low as 0.007 mM prevented resuscitation and a prolonged exposure to hydrogen peroxide at concentrations far under those inhibiting the growth of log-phase cells permanently damaged VBNC cells, which could not be resuscitated. However, the potential of culturability of VBNC cells could be preserved, at least for a part of the population, by plating the cells in the presence of catalase. The study also showed that during the resuscitation process, the cells gradually increased their resistance to hydrogen peroxide.ConclusionsThe timing and mode of induction of the VBNC state, as well as cell resuscitation and response to hydrogen peroxide, differed among Vibrio strains, indicating that induction and resuscitation from dormancy could vary in the context of species belonging to a single genus.

A novel technique of amniotic membrane suturing for ex vivo cultivated limbal epithelial stem cell expansion increases the number of progenitor cells

AbstractPurpose: Human limbal epithelial stem cells (HLESCs) reside in limbal epithelial crypts and continuously compensate for the loss of cells in the corneal epithelium. The lack or malfunction of HLESCs leads to a severe ocular surface disease called limbal epithelial stem cell deficiency (LSCD). Cultivated limbal epithelial stem cell transplantation (CLET) of expanded autologous limbal tissue on a human amniotic membrane (HAM) carrier is one of the most common methods for monocular LSCD. We hereby present a novel suturing technique of HAM prior to limbal biopsy expansion and cultivation that mimics natural limbal crypts.Methods: HLESCs derived from limbal biopsies were cultured ex vivo in complex media (COM) on flat or crypt‐like sutured HAMs for 3 weeks. The samples were then processed for immunohistochemistry (IHC) and analysed for progenitor markers: p63α and SOX9; markers for quiescence: CEBPD; proliferation: Ki‐67; differentiation: CX43 and CK3/12; transmembrane markers: E‐cadherin and N‐cadherin. The positivity of markers was counted and compared in cells cultivated on flat vs. crypt‐like sutured HAMs.Results: HLESCs cultured on folded, crypt‐like sutured HAMs had significantly more positivity for p63α, SOX9 (progenitor markers) and Ki‐67 (proliferation marker) compared to those cultured on flat HAMs. Fewer cells were positive for CK3/12 (differentiation marker) in cultures on folded compared to flat HAMs. The number of cells positive for CEBPD (quiescence marker), CX43 and E‐cadherin (differentiation markers) were equal in both groups (flat vs. folded HAMs).Conclusions: A novel technique of HAM suturing that forms crypt‐like undulations mimicking limbal epithelial crypts increases the number of early progenitor cells and could hence increase the quality of the ex vivo expanded limbal epithelial tissue and success for transplantation.

Amniotic membrane (AM) for ocular surface pathology: The old and the new

AbstractThe first amniotic membrane use was dated in 1910 as a skin graft substitute material. In the ophthalmological field, it was first used in the 1940 s as a conjunctival substitute after removal of fibrotic tissue, with good results when it was contracted with rabbit peritoneum. The procedure lost interest because the tissue could only be used fresh. Clinicians regained interest in the early 1990 s when new preservation, storage and distribution methods were already available.Application methods: The way to use the amniotic membrane depends on the pathological situation for which it is indicated. The depth, shape and size of the recipient area also play a role in the therapeutic decision.In general, there are three possible situations:An inlay transplantation as a graft: The AM works as a replacement of the lost tissue because it is remodelled into the host corneal matrix over time. The amnion is placed epithelial side up, as the membrane is able to act as a substrate for epithelial regeneration.An onlay transplantation as a patch: The transplant acts as a temporary biological bandage. The AM and the molecules that produces, protect the cornea from environmental aggressions, and it also has antifibrotic effect. The amnion is placed epithelial side down. Usually the product can be removed, although it is usually self‐degraded.Combinatorial transplantation as a sandwich: Two layers are transplanted, the deepest is placed epithelial side up and the other epithelial side down, obtaining the benefits of the two techniques.The graft provides structural integrity and the patch gives protection to de the graft, while the epithelium is expected to grow between the two layers.From our point of view this is the best option.Main indications as a graft:‐Corneal ulcerations and persistent epithelial defects.‐Limbal Stem Cells Deficiency (LSCD). It can be used in Simple Limbal Epithelial Transplantation (SLET), in Amnion‐Assisted Epithelial Redirection (ACER), and as a matrix for Cultivated Stem Cell Transplantation.‐In Glaucoma, to treat leaks from blebs.‐In Neoplasias, AM is used to act as a substrate for conjunctival migration and reconstruction following excision of both malignant and benign tumours.‐In Oculoplastic surgery, AM has been suggested as a potential alternative to mucosal membrane transplantation in lid revision, orbital linings or symblepharolysis.‐In Pterygium surgery, mainly in relapsing cases in which a very large resection of the conjunctiva is made.‐In Strabismus surgery, either to cover defects or to reduce muscle fibrosis and adhesion.Main indications as a patch, for Ocular Surface Wound Healing:‐In the acute stage of Chemical/Thermal injuries.‐In the acute stage of Cicatrising Conjunctivitis (Erythema Multiforme Diseases, Pemphigoid, Graft versus Hosts Disease).‐In recurrent corneal erosions.The potential risks of amniotic transplantation will be discussed.

Antibacterial activity against enterovirulent Escherichia coli strains from Bacillus amyloliquefaciens B31 and Bacillus subtilis subsp. subtilis C4: MALDI-TOF MS profiling and MALDI TOF/TOF MS structural analysis on lipopeptides mixtures.

In this work, two Bacillus strains isolated from honey (Bacillus subtilis subsp. subtilis C4; access code HQ828992) and from a waste of an artisanal tannery (Bacillus amyloliquefaciens B31; access code KP893752) were evaluated in order to determine their antibacterial activity against five enteropathogenic Escherichia coli strains. The number of viable cultivable cells of the different strains of E.coli analyzed was determined by plate count. The crude cell-free supernatants of both Bacillus strains exerted anti-E.coli activities, whereas only the lipopeptide fraction of B31 had significant E.coli inhibition. The lipopeptides produced by the Bacillus were analyzed using matrix-assisted laser desorption-ionization mass spectrometry (MALDI MS). The analysis was conducted combining the profiles (fingerprints) of the lipopeptides mixture and the individual lipopeptide fragmentation (tandem mass spectrometry [MS/MS] mode), both obtained from the same lipopeptides mixture sample, for higher output. Data obtained from C4 and B31 revealed that surfactin homologues were the most abundant lipopeptides produced by both strains studied. Additionally, kurstakin, iturin, and fengycin homologues were detected. Using the MS/MS mode, it was demonstrated that isobar compounds belonging to different families were produced by each Bacillus strain (e.g., C-16 bacillomycin D was detected in B31 samples, meanwhile C-15 iturin C was detected in C4). MS/MS analysis contributed with relevant information about the type of lipopeptides synthesized by Bacillus strains studied in this work.

Assessment of the Antibiofilm Performance of Chitosan-Based Surfaces in Marine Environments

Marine biofouling is a natural process often associated with biofilm formation on submerged surfaces, creating a massive economic and ecological burden. Although several antifouling paints have been used to prevent biofouling, growing ecological concerns emphasize the need to develop new and environmentally friendly antifouling approaches such as bio-based coatings. Chitosan (CS) is a natural polymer that has been widely used due to its outstanding biological properties, including non-toxicity and antimicrobial activity. This work aims to produce and characterize poly (lactic acid) (PLA)-CS surfaces with CS of different molecular weight (Mw) at different concentrations for application in marine paints. Loligo opalescens pens, a waste from the fishery industry, were used as a CS source. The antimicrobial activity of the CS and CS-functionalized surfaces was assessed against Cobetia marina, a model proteobacterium for marine biofouling. Results demonstrate that CS targets the bacterial cell membrane, and PLA-CS surfaces were able to reduce the number of culturable cells up to 68% compared to control, with this activity dependent on CS Mw. The antifouling performance was corroborated by Optical Coherence Tomography since PLA-CS surfaces reduced the biofilm thickness by up to 36%, as well as the percentage and size of biofilm empty spaces. Overall, CS coatings showed to be a promising approach to reducing biofouling in marine environments mimicked in this work, contributing to the valorization of fishing waste and encouraging further research on this topic.

Improving suppressive activity of compost on phytopathogenic microbes by inoculation of antagonistic microorganisms for secondary fermentation

Antimicrobial activity contributes to plant disease control property of composts but its source is still not clear. From composting cow manure during secondary fermentation, 50 microbial strains with antifungal activity were isolated and identified. Two bacterial strains Bacillus mojavensis B282 and Pseudomonas aeruginosa F288, antagonistic against both phytopathogenic fungi and bacteria, were respectively used as the inoculum of compost for secondary fermentation. Inoculation of B282 or F288 significantly shifted microbial community structure of compost and genera functionally linked to antagonistic activity and plant growth promotion were enriched. Notably, culturable cells of B282 increased by about 40 times during secondary fermentation. The inoculation of each strain significantly increased antifungal activity of compost extracts and enhanced disease suppressive effects of compost on wheat root rot. This study demonstrates that inoculation of compost-indigenous microorganisms could improve antimicrobial activity of compost and provides a low-cost strategy for producing bio-organic fertilizers with biocontrol function.

Assessment of shelf-life and metabolic viability of a multi-strain synbiotic using standard and innovative enumeration technologies.

The number of live bacterial cells is the most used parameter to assess the quality of finished probiotic products. Plate counting (PC) is the standard method in industry to enumerate cells. Application of PC implies critical aspects related to the selection of optimal nutrient media and growth conditions and underestimation of viable but not cultivable (VBNC) cells. Flow-cytometry (FC) is a culture-independent methodology having the potential to selectively enumerate live, damaged, and dead cells representing a powerful tool for in-depth monitoring of probiotic products. We monitored the shelf life of a clinical batch of a synbiotic composition PDS-08 targeting the pediatric population by means of PC and FC according to International Conference on Harmonization (ICH) pharma guidelines testing the Arrhenius model as predictive tool; PC enumeration revealed higher destruction rate than FC suggesting a faster reduction in cultivability than membrane integrity and thus a possible shift of the bacteria into a VBNC status. PDS-08 maintained acidification capability over time, when re-suspended in nutrient medium, even in samples tested sub-optimally for CFU detection (below 1 billion cells/dose). Due to similar kinetics described by the study of metabolic activity and membrane integrity, FC might be suggested as a valid tool for the study of functional stability of a probiotic product.

Carbon nanotube-based surfaces: Effect on the inhibition of single- and dual-species biofilms of Escherichia coli and Enterococcus faecalis

This study aimed to investigate the influence of multi-walled carbon nanotube (MWCNT) loading on the antibiofilm activity of MWCNT/poly(dimethylsiloxane) (PDMS) composites against single- and dual-species biofilms of Escherichia coli and Enterococcus faecalis. MWCNT/PDMS composites containing different MWCNT loadings (1, 2, and 3 wt%) were synthesized and tested for their ability to reduce biofilm formation. Surfaces were evaluated using a parallel plate flow chamber for 24 h under the hydrodynamic conditions prevailing in urinary catheters. Biofilm formation by E. coli and E. faecalis was assessed by determining the number of total, viable and culturable cells, and the biofilm architecture was analyzed by confocal laser scanning microscopy (CLSM). Results revealed that the composite with the highest MWCNT loading (3%) was the most effective against E. coli and E. faecalis biofilms. When compared with bare PDMS, it significantly reduced the number of total (63%), viable (74%), and culturable (77%) cells of E. faecalis biofilms, and the number of viable (50%) and culturable (64%) cells of E. coli biofilms. The effect of these MWCNT-based composites on the inhibition of biofilm formation was corroborated by CLSM, where reductions of up to 57% and 64% in biofilm biovolume and thickness, respectively, were observed. Even though dual-species biofilms presented lower susceptibility to the antimicrobial activity of MWCNT/PDMS composites, a reduction of 32% in the number of culturable cells was obtained with 3% MWCNT/PDMS surfaces. Taken together, these results suggest that MWCNT/PDMS composites are promising materials for developing novel antimicrobial and antifouling coatings for urinary tract devices.

Deciphering the induction of Listeria monocytogenes into sublethal injury using fluorescence microscopy and RT-qPCR

The adaptive response of bacterial cells to changing environmental conditions depends on the behavior of single cells within the population. Exposure of Listeria monocytogenes to sublethal acidic conditions in foods or in the gastrointestinal track of the host may induce injuries relevant to difficult physiological states within the dormancy continuum. In this study, exposure to acidic conditions (acetic-AA and hydrochloric acid-HCl adjusted to pH 3.0, 2.7, 2.5 at 20 °C for 5 h) was used to evaluate injury of L. monocytogenes, Scott A strain. To differentiate the resistant sub-population from the total, Tryptic Soy Agar with 0.6 % Yeast Extract (TSAYE) supplemented or not with 5 % NaCl were comparatively used. Sublethally injured cells were detected by comparing plate counts with fluorescence microscopy, using combinations of CFDA (viability) and Propidium-Iodide (death). Effect of acid stress on the relative transcription of clpP, mazE, mazF, relA, gadC, gadD, gadB, sigB, inlA and prfA upon transition of total population into different physiological stages was evaluated through RT-qPCR. AA treated cells showed measurable logarithmic reduction at pH 2.7 and 2.5, while there was a significant percentage of CFDA−/PI+ cells. Evaluation of the potentially culturable population on TSAYE, from the percentage of CFDA/PI-stained cells, revealed that unstained cells represented a non-culturable sub-population. Exposure to Ringer's solution pH 2.7, adjusted with AA, resulted in higher percentages of non-esterase active with membrane integrity cells (CFDA−/PI−) compared to the percentages of the enumerated culturable cells on TSAYE after 4 and 5 h. Under the same conditions, after 1 h of exposure macroscopic observation revealed size colony variations (SCVs) of the total population (CFU on TSAYE). L. monocytogenes retained its culturability after hydrochloric acid exposure, while cells remained metabolically active (CFDA+). However, a stochastic change in cell's shape, was detected after exposure to pH 3.0 and 2.5, adjusted with HCl, for 2 h at 20 °C. A pattern of gene up-regulation was observed during treatment with AA pH 2.7 and HCl pH 3.0 at the 3rd h of exposure. Deciphering L. monocytogenes sublethal injury sheds light into the physiological and molecular characteristics of this state and provides the food science community with quantitative data to improve risk assessment.

Survival virulent characteristics and transcriptomic analyses of Vibrio mimicus exposed to starvation

Vibrio mimicus is a global causative agent of vibriosis in a variety of aquatic animals and causes major economic losses to aquaculture. It could survive in water for extended periods of time under environmental stress, but its survival strategy remains unknow. This study described the survival, virulent and gene expression changes of V. mimicus cells undergoing starvation stress. After 4 weeks’ cultivation in media without nutrients, V. mimicus Y4 showed reduced rates of activity with marked changes in morphology and physiological activities. The culturable cell counts declined gradually to 104 CFU/mL and the shape changed from rod-shaped to coccoid with short rods or spherical. The motility of starved cells decreased after starvation and the biofilm production was significantly lower than wild cells. The starved cells still produced β-hemolysis, lecithinase and caseinase, but its infectivity to Macrobrachium nipponense was weakened. To investigate the mechanism behind morphological and physiological changes, we further analyzed differently expressed genes (DEGs) between starved and wild cells at the whole transcriptional level. The RNA-seq analysis demonstrated that large-scale DEGs were involved in transferase, membrane, dehydrogenase, synthase, flagellar, hemolysin, pilus assembly, and starvation, etc. Among them, the well-known virulence-related genes were downregulated significantly, including vmh, pilA, vipA, capB, tadC, huvX, ompA, etc. These data provide a key resource to understand the regulatory mechanisms of V. mimicus to starvation stress.

Cross-contamination of Escherichia coli O157:H7 and Listeria monocytogenes in the viable but non-culturable (VBNC) state during washing of leafy greens and the revival during shelf-life

Some water disinfection treatments, such as chlorine and chlorine dioxide, used in the fresh-cut industry to maintain the microbiological quality of process water (PW), inactivate bacterial cells in the water but they also lead to the induction of an intermediate state between viable and non-viable known as viable but non-culturable (VBNC) state. Viable cells can participate in cross-contamination events but the significance of VBNC cells in PW, transfer to the product and potential resuscitation capacity during storage is unclear. The present study aims to determine first, if VBNC cells present in PW can cross-contaminate leafy greens during washing and secondly its potential revival during shelf-life. Process water characterized by a high chemical oxygen demand, due to the presence of high levels of organic matter, was inoculated with Listeria monocytogenes or Escherichia coli O157:H7. Inoculated PW was then treated for 1 min with chlorine dioxide (3 mg/L) or chlorine (5 mg/L) to generate VBNC cells. Absence of culturable cells was confirmed by plate count and VBNC cells by viability quantitative polymerase chain reaction (v-qPCR) complemented with two dyes, ethidium (EMA) and propidium (PMAxx) monoazide. Cross-contamination of shredded lettuce was demonstrated by monitoring the VBNC cells after washing the product for 1 min in the contaminated PW and during shelf life (15 days at 7 °C). In the case of L. monocytogenes, considering the total concentration of L. monocytogenes VBNC cells present in the PW, only a low proportion of cells were able to cross-contaminate the product during washing. VBNC L. monocytogenes cells were able to resuscitate on the product during shelf life, although levels of cultivable bacteria, close to the limit if detection (0.7 ± 0.0 log CFU/g), were only detected at the end of storage. On the other hand, VBNC cells of E. coli O157:H7 present in PW were not able to cross-contaminate shredded lettuce during washing. Moreover, when shredded lettuce was artificially inoculated with VBNC E. coli O157:H7, resuscitation of the VBNC cells during storage (15 days at 7 °C) was not observed. Based on the results obtained, injured L. monocytogenes cells present in the PW are able to be transferred to the product during washing. If VBNC L. monocytogenes cells present in leafy greens (shredded lettuce and baby spinach), they can resuscitate, although cultivable numbers remained very low. Taking all the results together, it could be concluded that under industrial conditions, VBNC cells can be transferred from water to product during washing, but their capacity to resuscitate in the leafy greens during storage is low.

Acinetobacter baylyi Strain BD413 Can Acquire an Antibiotic Resistance Gene by Natural Transformation on Lettuce Phylloplane and Enter the Endosphere.

Antibiotic resistance spread must be considered in a holistic framework which comprises the agri-food ecosystems, where plants can be considered a bridge connecting water and soil habitats with the human microbiome. However, the study of horizontal gene transfer events within the plant microbiome is still overlooked. Here, the environmental strain Acinetobacter baylyi BD413 was used to study the acquisition of extracellular DNA (exDNA) carrying an antibiotic resistance gene (ARG) on lettuce phylloplane, performing experiments at conditions (i.e., plasmid quantities) mimicking those that can be found in a water reuse scenario. Moreover, we assessed how the presence of a surfactant, a co-formulant widely used in agriculture, affected exDNA entry in bacteria and plant tissues, besides the penetration and survival of bacteria into the leaf endosphere. Natural transformation frequency in planta was comparable to that occurring under optimal conditions (i.e., temperature, nutrient provision, and absence of microbial competitors), representing an entrance pathway of ARGs into an epiphytic bacterium able to penetrate the endosphere of a leafy vegetable. The presence of the surfactant determined a higher presence of culturable transformant cells in the leaf tissues but did not significantly increase exDNA entry in A. baylyi BD413 cells and lettuce leaves. More research on HGT (Horizontal Gene Transfer) mechanisms in planta should be performed to obtain experimental data on produce safety in terms of antibiotic resistance.

Pre-Training on In Vitro and Fine-Tuning on Patient-Derived Data Improves Deep Neural Networks for Anti-Cancer Drug-Sensitivity Prediction.

Simple SummaryCancer cell lines vary greatly from one another because each one underwent a combination of random mutations. Therefore, the effectiveness of anti-cancer drugs also varies across cancer cell lines. In order to understand which drugs are effective against which cancer cells, researchers expose cultivated cell lines to drug candidates. However, these results are not entirely realistic, mostly because cultivated cells live in a two-dimensional in vitro environment without interacting with other types of cells. There are more realistic ways to test drug effectiveness—e.g., organoids that are 3D-printed from tumor cells—but since these processes are more complex, much fewer data are available. We studied an approach in which a neural network is first trained on the wealth of available in vitro drug-sensitivity data, before being fine-tuned on smaller but more realistic databases. We found that this training procedure improves the neural network’s ability to predict how effective a particular drug will be for a given tumor cell line. Such neural networks can serve as a tool, both for personalized treatment of cancer and for drug development.Large-scale databases that report the inhibitory capacities of many combinations of candidate drug compounds and cultivated cancer cell lines have driven the development of preclinical drug-sensitivity models based on machine learning. However, cultivated cell lines have devolved from human cancer cells over years or even decades under selective pressure in culture conditions. Moreover, models that have been trained on in vitro data cannot account for interactions with other types of cells. Drug-response data that are based on patient-derived cell cultures, xenografts, and organoids, on the other hand, are not available in the quantities that are needed to train high-capacity machine-learning models. We found that pre-training deep neural network models of drug sensitivity on in vitro drug-sensitivity databases before fine-tuning the model parameters on patient-derived data improves the models’ accuracy and improves the biological plausibility of the features, compared to training only on patient-derived data. From our experiments, we can conclude that pre-trained models outperform models that have been trained on the target domains in the vast majority of cases.

Bactericidal and anti-biofilm effects of uncharged and cationic ultrasound-responsive nitric oxide microbubbles on Pseudomonas aeruginosa biofilms

Bacterial biofilms are a major and ongoing concern for public health, featuring both inherited genetic resistance traits and a conferred innate tolerance to traditional antibiotic therapies. Consequently, there is a growing need for novel methods of drug delivery, to increase the efficacy of antimicrobial agents. This research evaluated the anti-biofilm and bactericidal effects of ultrasound responsive gas-microbubbles (MBs) of either air or nitric oxide, using an in vitro Pseudomonas aeruginosa biofilm model grown in artificial wound medium. The four lipid-based MB formulations evaluated were room-air MBs (RAMBs) and nitric oxide MBs (NOMBs) with no electrical charge, as well as cationic (+) RAMBs+ and NOMBs+. Two principal treatment conditions were used: i) ultrasound stimulated MBs only, and ii) ultrasound stimulated MBs with a sub-inhibitory concentration (4 µg/mL) of the antibiotic gentamicin. The total treatment time was divided into a 60 second passive MB interaction period prior to 40 second ultrasound exposure; each MB formulation was tested in triplicate. Ultrasound stimulated RAMBs and NOMBs without antibiotic achieved reductions in biofilm biomass of 93.3% and 94.0%, respectively. Their bactericidal efficacy however was limited, with a reduction in culturable cells of 26.9% and 65.3%, respectively. NOMBs with sub-inhibitory antibiotic produced the most significant reduction in biofilm biomass, corresponding to a 99.9% (SD ± 5.21%); and a 99.9% (SD ± 0.07%) (3-log) reduction in culturable bacterial cells. Cationic MBs were initially manufactured to promote binding of MBs to negatively charged biofilms, but these formulations also demonstrated intrinsic bactericidal properties. In the absence of antibiotic, the bactericidal efficacy of RAMB+ and NOMB+ was greater that of uncharged counterparts, reducing culturable cells by 84.7% and 86.1% respectively; increasing to 99.8% when combined with antibiotic. This study thus demonstrates the anti-biofilm and bactericidal utility of ultrasound stimulated MBs, and specifically is the first to demonstrate the efficacy of a NOMB for the dispersal and potentiation of antibiotics against bacterial biofilms in vitro. Importantly the biofilm system and complex growth-medium were selected to recapitulate key morphological features of in vivo biofilms. The results us offer new insight for the development of new clinical treatments, for example, in chronic wounds.

Abundance, isolation, and characterization of halotolerant microbes from common oligosaline soils

Halotolerant microbes that grow at high NaCl concentrations were studied in common soils with low salinity. The abundance of microbes in common oligosaline (<1.0% salinity) soils was measured by most probable number analyses, where statistically valid replicates of serial dilutions were assessed for growth in liquid Salt Plains medium with 0.1, 10, or 20% (w/v) NaCl. Common Kansas soils were collected from oligosaline agricultural fields (corn, cotton, sorghum, and soybean), pond margin, prairie grassland, and woodland. The abundance of microbes able to grow at 10% NaCl was 0.0025–0.32% of the total counts at 0.1% NaCl. Only marginal growth was observed at 20% NaCl, about 100-fold lower than at 10% NaCl. Bacterial isolates were collected from 20% NaCl enrichments of oligosaline turf soil by repetitive streak-plating. The bacteria were identified by 16S rRNA gene sequencing and characterized. The isolates clustered with low G+C Gram-positive genera (Bacillus, Halobacillus, Staphylococcus, and Virgibacillus). Broad halotolerances (0.1–20% NaCl) were observed for most isolates, but all grew at low salt. Soils were boiled to kill vegetative cells and compared by most probable number analyses with unboiled specimens. The abundance of microbes able to grow at 10% NaCl showed a 3-log decrease with boiling, while microbes able to grow at 0.1% NaCl showed a 5-log decrease in abundance with boiling. It appears that halotolerant microbes in oligosaline soils are cultivable vegetative cells, not perennating structures such as spores. Discrete salt phases are not obvious in oligosaline soils, but could provide a selective advantage to autochthonous halotolerant microbes. Allochthonous microbes associated with salt evaporite particles may be transported by winds, thereby dispersing deliquescent hypersaline microhabitats.