Planktonic State Research Articles

Comparison of the Bacterial Inactivation Efficiency of Water Activated by a Plasma Jet Source and a Pin-to-Pin Electrode Configuration Source

In this comparative study, the bacterial inactivation efficiency of plasma-activated water (PAW) generated by two distinct plasma reactors, one utilizing a nitrogen plasma jet electrode and the other a hybrid argon plasma reactor, was explored. The present study involved the assessment of antimicrobial activity against suspensions of three Gram-positive and three Gram-negative bacterial strains in their planktonic cell state. Bacterial suspensions were introduced into PAW five days after generation. Subsequently, the viability of the bacteria was assessed at various time intervals, specifically at 0.5, 1, 3, 5, 10, and 24 h, in order to evaluate the effect of inactivation. Structural changes in bacteria after PAW treatment were assessed using a scanning electron microscope (SEM). The physicochemical properties of PAW, including pH, conductivity, and concentrations of H2O2, NO2−, and NO3− during aging were measured. The present study demonstrated the effective inactivation of the tested bacterial strains by PAW. Gram-positive bacteria displayed greater resistance compared to Gram-negative species, with the lowest reductions in bacterial counts observed for B. cereus, and the highest for Escherichia coli O157:H7. Morphological damage was evident across all bacterial species examined. Physicochemical measurements showed slow decay of the reactive species in the aging process. This study illustrated the potential utility of PAW as an alternative disinfectant.

Planktonic and biofilm states of Staphylococcus aureus isolated from bone and joint infections and the in vitro effect of orally available antibiotics.

To demonstrate the in vitro activity of orally available antibiotics against Staphylococcus aureus isolated from bone or orthopedic implant materials. The biofilm eradication of the combination of three antibiotics was also assessed. Clinical isolates from orthopedic infection samples were collected, and S. aureus isolates were classified according to their biofilm production and composition. Almost all S. aureus isolates (n=36, 97.3%) produced biofilm and the major biofilm components were polysaccharides. Antimicrobial susceptibility was determined in planktonic (minimal inhibitory concentration; MIC) and biofilm cells (minimal biofilm eradication concentration; MBEC) using the MBEC Calgary Device. Overall, the MBEC ranged higher than the MIC. When combined at borderline-susceptible concentrations, moxifloxacin-rifampin and doxycycline-rifampin were both able to eradicate biofilms in a third of the strains whereas the doxycycline-moxifloxacin combination proved ineffective at eradicating biofilm, inhibiting it only in three strains. We propose rifampin in combination with moxifloxacin or doxycycline for the design of clinical trials of bone and/or orthopedic device infection without proper debridement or material retention.

Biased eviction of variant histone H3 nucleosomes triggers biofilm growth in Candida albicans.

Candida albicans lives as a commensal in most healthy humans but can cause superficial skin infections to life-threatening systemic infections. C. albicans also forms biofilms on biotic and abiotic surfaces. Biofilm cells are difficult to treat and highly resistant to antifungals. A specific set of genes is differentially regulated in biofilm cells as compared to free-floating planktonic cells of C. albicans. In this study, we addressed how a variant histone H3VCTG, a previously identified negative regulator of biofilm formation, modulates gene expression changes. By providing compelling evidence, we show that biased eviction of H3VCTG nucleosomes at the promoters of biofilm-relevant genes facilitates the accessibility of both transcription activators and repressors to modulate gene expression. Our study is a comprehensive investigation of genome-wide nucleosome occupancy in both planktonic and biofilm states, which reveals transition to an open chromatin landscape during biofilm mode of growth in C. albicans, a medically relevant pathogen.

Воздействие бовгиалуронидазы азоксимера на бактериальные биопленки в эякуляте пациентов с хроническим простатитом.

Introduction. The use of molecular biological methods in the analysis of bacterial diversity (metataxonomics) of prostate secretion and ejaculate to assess the male microbiota made it possible to identify the bacterial diversity of the microbiota in chronic prostatitis. At the same time, many strains of uropathogens identified in chronic prostatitis show the ability to form biofilms, while there are no generally accepted methods for treating infections caused by bacterial biofilms. Purpose of the study. To study the effect of bovhyaluronidase azoximer in chronic prostatitis, which may be due to the enzymatic action of hyaluronidase on the matrix of bacterial biofilms, which increases the bioavailability of antibiotics, leading to the transition of bacteria into a planktonic state. Material and methods. An electron microscopic study of the ejaculate of 42 patients with chronic prostatitis was performed, of which 32 randomly selected patients received combined antibiotic treatment (4-6 weeks) together with bovhyaluronidase azoxymer (12 weeks) (group I), 10 patients received only antibiotic therapy according to clinical guidelines (group II). On ultrathin sections, the number of bacterial microcolonies and neutrophilic leukocytes per 100 spermatozoa was counted. Results. In the semen of 30 patients, bacterial biofilms with a characteristic morphology were detected: the presence of heterogeneous microorganisms in one microcolony and the presence of a fibrous matrix in which bacteria are immersed. After therapy, in group I, a decrease in the number of bacterial microcolonies, the disappearance of the extracellular matrix, and isolated single bacteria (planktonic state) were detected, in some cases, damage to the bacterial cell wall was detected. In group II quantitative changes in the content of microcolonies and neutrophilic leukocytes were also revealed, however, the morphology of microcolonies in group II did not change. Conclusions. electron microscopy in the study of the ejaculate of patients with chronic prostatitis allows us to reveal the features of the structure of bacterial biofilms. In combined antibiotic therapy together with bovhyaluronidase azoxymer, the biofilm matrix changes and the bacteria transform into a planktonic form.

A Sensor for Monitoring the Antimicrobial Activity of Wound Dressings for Both Surgical Site Infections (SSIs) and Chronic Wounds.

Antimicrobial impregnated wound dressings are a critical tool for the management, prevention, and control of surgical site infections (SSIs) and infected chronic wounds. However, the sustained therapeutic antimicrobial activity of the dressing when employed for extended periods cannot be readily determined in vivo. Consequently, dressings are changed frequently to ensure that their antimicrobial activity is maintained. Whilst frequent dressing changes allow the wound to be assessed, this is time-consuming and can cause disruption to the wound bed impairing the healing process. Furthermore, this increases medical costs for the patient and hospitals. This paper introduces a novel concept to monitor the therapeutic levels of an antimicrobial component within a wound dressing ensuring the wound dressing remains "fit for purpose" and avoiding indiscriminate use of antiseptics. This could help to inform clinicians whether the antimicrobial is still being delivered at therapeutic levels and as such when to change the dressing ensuring timely positive clinical outcomes. Silver has been used historically as an antimicrobial agent and is ubiquitous in current generations of antimicrobial wound dressings. However, its activity is complex due to the poor solubility of silver ions in the presence of chloride and the effect of complexation by other components in the dressing and wound ecosystem, not least by serum proteins. In this paper, we detail an electrochemical silver sensor (5D patent protected - WO2023275553A1), constructed using a platinum (Pt) nanoband array electrode, and characterise its response to silver ions. This is determined in the presence of bovine serum albumin (BSA) and simulated wound fluid (SWF) containing chloride and rationalised using atomic analysis of the composition of the SWF. The sensor response in SWF is compared with the antimicrobial activity of silver against Pseudomonas aeruginosa in the planktonic and biofilm state, as a function of the amount of silver nitrate added. At low concentrations, silver in SWF has good solubility but reduced antimicrobial effect due to binding of silver by BSA as shown by the sensor response. At intermediate concentrations, above 10ppm, the silver was efficacious on both planktonic microorganisms and biofilm impregnated with microorganisms and readily detected with the sensor. At high concentrations, silver precipitates and both the silver in solution and the sensor response plateaus. The data demonstrates how the sensor correlates with the antimicrobial activity of the silver in vitro and how this could be used to actively monitor antimicrobials in vivo.

Examining the role of glycoside hydrolases in local rheology of Pseudomonas aeruginosa biofilms

Current research strategies in the treatment of biofilm infections have focused on dispersal, in which bacteria are made to vacate the extracellular polymeric substance (EPS) surrounding them and return to a planktonic state where antimicrobial treatments are more effective. Glycoside hydrolases (GHs), which cleave bonds in EPS polysaccharides, have been shown to promote dispersal in Pseudomonas aeruginosa biofilms. The dispersal mechanism is possibly due to GHs’ ability to directly release bacteria from the EPS, disrupt EPS’ ability to regulate the environment, or reduce overall mechanical stability. In this work, passive microrheology is used to examine the relevance of the last mechanism by exploring the effects of three GHs (α-amylase, cellulase, and xylanase) known to disperse P. aeruginosa on local biofilm viscoelasticity. Compared to control studies in wild-type strains, it is found that treatment with all three GHs results in statistically relatively less elastic and stiffer biofilms, indicating that changes to mechanical stability may be a factor in effective dispersal. Both cellulase and xylanase were observed to have the greatest impact in creating a less stiff and elastic biofilm; these GHs have been observed to be effective at dispersal in the published results. Each GH was further tested on biofilms grown with strains that produced EPS missing specific polysaccharide components. Cellulase specifically targeted Psl, which forms the major structural and mechanical backbone of the EPS, explaining its efficacy in dispersal. However, xylanase did not appear to exhibit any affinity to any polysaccharide within the EPS based on the microrheology results. Overall, these results suggest that the local microrheology of the biofilms is impacted by GHs and that may be one of the factors that is causing the ability of these therapeutics to enhance dispersal.

Plasma-activated water for inactivation of Salmonella Typhimurium avirulent surrogate: Applications in produce and shell egg and understanding the modes of action

Plasma-activated water (PAW) is gaining interest as a novel sanitizer for foods. However, the sanitizing efficacy of PAW varies under different conditions, and its modes of action and impacts on food quality need further investigation for it to be applied in the food industry. This study evaluated the inactivation efficacy of PAW against a Salmonella Typhimurium avirulent surrogate, investigated the underlying mechanisms, and assessed the impacts of PAW on the safety and quality of cherry tomatoes and shell eggs. PAW was effective in inactivating S. Typhimurium in a planktonic state (1.5–5.8 log CFU reduction in 2–5 min) and on the food surface (>5 log CFU reduction in 5 min). PAW prepared with deionized water vs. tap water did not differ significantly in sanitizing efficacy. Various reactive oxygen and nitrogen species (RONS) contributed to the antimicrobial property of PAW, with nitric oxide playing the most significant role. The acidic nature of PAW might synergistically affect bacteria by disrupting cell membrane and enhancing RONS permeation. PAW did not modify the visual appearance and internal pH of the treated foods. This study demonstrates that PAW has the potential to be applied as a novel sanitizer for fresh produce and egg washing.

Germicidal effect of intense pulsed light on Pseudomonas aeruginosa in food processing.

Pseudomonas aeruginosa (P. aeruginosa) can cause serious infections in many parts of the body and is also an underestimated foodborne pathogen. Intense pulsed light sterilization is recognized for its high sterilization efficiency, flexible and safe operation and ease of installation on production lines, which makes up for the shortcomings of several other physical sterilization technologies. This experiment studied the killing efficiency of different capacitances (650 μF, 470 μF, and 220 μF) of intense pulsed light on foodborne pathogenic microorganisms P. aeruginosa in the models of liquid food models, 96-well cell plates, and polycarbonate membrane models at room temperature (25°C) and refrigerated (4°C) environments to provide data to support the application of IPL sterilization devices in food processing. The IPL was very effective in killing P. aeruginosa in the planktonic state as well as in the early and mature biofilm states, meeting target kill rates of 100%, 99.99%, and 94.33% for a given number of exposures. The biofilms formed in the polycarbonate membrane model and the 96-well plate model were more resistant to killing compared to the planktonic state. To achieve the same bactericidal effect, the number of flashes increased with decreasing capacitance. The bactericidal effect of IPL on P. aeruginosa was significantly influenced by the state of the bacterium. The larger the capacitance the higher the number of pulses and the better the sterilization effect on P. aeruginosa.

Repurposing anti-inflammatory drugs for fighting planktonic and biofilm growth. New carbazole derivatives based on the NSAID carprofen: synthesis, in silico and in vitro bioevaluation.

One of the promising leads for the rapid discovery of alternative antimicrobial agents is to repurpose other drugs, such as nonsteroidal anti-inflammatory agents (NSAIDs) for fighting bacterial infections and antimicrobial resistance. A series of new carbazole derivatives based on the readily available anti-inflammatory drug carprofen has been obtained by nitration, halogenation and N-alkylation of carprofen and its esters. The structures of these carbazole compounds were assigned by NMR and IR spectroscopy. Regioselective electrophilic substitution by nitration and halogenation at the carbazole ring was assigned from H NMR spectra. The single crystal X-ray structures of two representative derivatives obtained by dibromination of carprofen, were also determined. The total antioxidant capacity (TAC) was measured using the DPPH method. The antimicrobial activity assay was performed using quantitative methods, allowing establishment of the minimal inhibitory/bactericidal/biofilm eradication concentrations (MIC/MBC/MBEC) on Gram-positive (Staphylococcus aureus, Enterococcus faecalis) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) strains. Computational assays have been performed to assess the drug- and lead-likeness, pharmacokinetics (ADME-Tox) and pharmacogenomics profiles. The crystal X-ray structures of 3,8-dibromocarprofen and its methyl ester have revealed significant differences in their supramolecular assemblies. The most active antioxidant compound was 1i, bearing one chlorine and two bromine atoms, as well as the CO2Me group. Among the tested derivatives, 1h bearing one chlorine and two bromine atoms has exhibited the widest antibacterial spectrum and the most intensive inhibitory activity, especially against the Gram-positive strains, in planktonic and biofilm growth state. The compounds 1a (bearing one chlorine, one NO2 and one CO2Me group) and 1i (bearing one chlorine, two bromine atoms and a CO2Me group) exhibited the best antibiofilm activity in the case of the P. aeruginosa strain. Moreover, these compounds comply with the drug-likeness rules, have good oral bioavailability and are not carcinogenic or mutagenic. The results demonstrate that these new carbazole derivatives have a molecular profile which deserves to be explored further for the development of novel antibacterial and antibiofilm agents.

Aminocoumarin derivatives grafted on graphene oxide - new antimicrobial agents to combat the resistance of Mycobacterium tuberculosis and ESKAPE pathogens

Two hybrid materials, based on 7-amino-4-(trifluoromethyl)coumarin and 7-amino-4-methyl-coumarin that attach covalently to graphene oxide (GO) have been synthesized in two steps: i) increasing the number of carboxyl groups required for functionalization with aminocoumarin derivatives by derivatization of the hydroxyls groups with chloroacetic acid, transforming GO into GO-COOH material; ii) activation of carboxylic groups using the carbodiimide-promoted reaction. The obtained composites were characterized by numerous methods that highlighted their successful obtaining. The antimicrobial activity was evaluated on resistant Mycobacterium tuberculosis strains, as well as on ESKAPE pathogens ((Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) in planktonic and biofilm growth states. The biocompatibility of the materials has been assayed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay as well as by measuring the level of nitric oxide/total reactive oxygen species and superoxide release in treated cells. The results have shown that materials exhibited improved inhibitory activity against resistant M. tuberculosis strains in comparison with GO, but also against planktonic and adherent strains. Also, the tested composites have been proved to be biocompatible on the MRC-5 fibroblast cells, demonstrating their promising potential to develop novel agents effective against multidrug resistant pathogens, including M. tuberculosis, as well as by the activation of a pro-inflammatory and of an oxidative response in the mammalian cells.

O01 Why is urinary tract infection so hard to diagnose and treat? Hints from a unique human microtissue model of bladder infection

Abstract Although urinary tract infection (UTI) appears, on the face of it, to be a simple bacterial infection, upwards of one in four UTI cases recur despite antibiotic therapy. While antimicrobial resistance after empirical treatment undoubtedly accounts for some of these failures, recurrent UTI despite the administration of an appropriate antibiotic, or in the face of prophylactic therapy, remains poorly understood and is likely due to complex and multifactorial aspects of the interaction between the pathogen and its host. In parallel, the role of the healthy uromicrobiome in this process is also far from clear. The vast majority of what little we know about UTI host/pathogen interactions at the urothelial surface has been extrapolated from forced infection experiments in mice, a species that does not naturally experience UTI and whose bladder environment differs significantly from that of humans. For this reason, our lab has been developing alternative, human-cell-based lab models to study the behaviour of both uropathogens and healthy commensals using fully stratified and differentiated urothelial platforms, including lab-on-chip models with flow and stretch parameters. In this talk, I will present recent data putting the most common uropathogens through their paces in our most advanced static model, 3D-UHU. All uropathogens tested, including Escherichia coli, Klebsiella, Enterococcus, Pseudomonas, Group B Streptococcus and Proteus, were able to invade the urothelium to take up residence inside cells, where they would be expected to avoid detection by diagnostic urine tests as well as host neutrophils. Intracellular residence would also foil most oral antibiotics, which are poorly permeant to the urothelial barrier. Some bacteria species, especially Pseudomonas and Proteus, formed biofilms on the surface of the urothelium, a behaviour that would also help the bacteria to withstand antibiotic treatment. What's more, we observed that some bacteria susceptible to antibiotics in diagnostic media were effectively resistant in the human organoid environment, suggesting that there could be a disconnect between predicted drug and clinical outcome. This phenomenon may be partly explained by our experiments showing that, in some species of uropathogen, the urine environment encourages biofilm formation over the free planktonic state. These experiments highlight diverse infection strategies, shed light on how diagnosis and treatment can fail, and reveal strategies for improving both in the future.

Nanodevices based on mesoporous glass nanoparticles enhanced with zinc and curcumin to fight infection and regenerate bone.

Nanotechnology-based approaches are emerging as promising strategies to treat different bone pathologies such as infection, osteoporosis or cancer. To this end, several types of nanoparticles are being investigated, including those based on mesoporous bioactive glasses (MGN) which exhibit exceptional structural and textural properties and whose biological behaviour can be improved by including therapeutic ions in their composition and loading them with biologically active substances. In this study, the bone regeneration capacity and antibacterial properties of MGNs in the SiO2-CaO-P2O5 system were evaluated before and after being supplemented with 2.5% or 4% ZnO and loaded with curcumin. in vitro studies with preosteoblastic cells and mesenchymal stem cells allowed determining the biocompatible MGNs concentrations range. Moreover, the bactericidal effect of MGNs with zinc and curcumin against S. aureus was demonstrated, as a significant reduction of bacterial growth was detected in both planktonic and sessile states and the degradation of a pre-formed bacterial biofilm in the presence of the nanoparticles also occurred. Finally, MC3T3-E1 preosteoblastic cells and S. aureus were co-cultured to investigate competitive colonisation between bacteria and cells in the presence of the MGNs. Preferential colonisation and survival of osteoblasts and effective inhibition of both bacterial adhesion and biofilm formation of S. aureus in the co-culture system were detected. Our study demonstrated the synergistic antibacterial effect of zinc ions combined with curcumin and the enhancement of the bone regeneration characteristics of MGNs containing zinc and curcumin to obtain systems capable of simultaneously promoting bone regeneration and controlling infection. STATEMENT OF SIGNIFICANCE: In search of a new approach to regenerate bone and fight infections, a nanodevice based on mesoporous SiO2-CaO-P2O5 glass nanoparticles enriched with Zn2+ ions and loaded with curcumin was designed. This study demonstrates the synergistic effect of the simultaneous presence of zinc ions and curcumin in the nanoparticles that significantly reduces the bacterial growth in planktonic state and is capable to degrade pre-formed S. aureus biofilms whereas the nanosystem exhibits a cytocompatible behaviour in the presence of preosteoblasts and mesenchymal stem cells. Based on these results, the designed nanocarrier represents a promising alternative for the treatment of acute and chronic infections in bone tissues, while avoiding the significant current problem of bacterial resistance to antibiotics.

Photodynamic Activity of Chlorophyllin and Polyethylenimine on Pseudomonas aeruginosa Planktonic, Biofilm and Persister Cells.

Antimicrobial photodynamic inactivation is considered a promising antimicrobial approach that may not develop resistance in the near future. Here, we investigate the influence of the photosensitizer chlorophyllin (CHL) and the cationic permeabilizer polyethylenimine (PEI), exposed to a red light-emitting diode, on the human pathogen Pseudomonas aeruginosa free-living planktonic cells, the sessile biofilm and persister cells. The broth microdilution checkerboard method was used to test antimicrobial susceptibility. As a substrate for biofilms, the Calgary biofilm device was used, and the quantification of the biofilm biomass was carried out using a crystal violet assay. Serine hydroxamate was used for the induction of persisters. Our findings reveal that PEI ameliorates the antimicrobial activity of CHL against P. aeruginosa planktonic and biofilm states, and the concentration required to eradicate the bacteria in the biofilm is more than fourfold that is required to eradicate planktonic cells. Interestingly, the persister cells are more susceptible to CHL/PEI (31.25/100 µg mL-1) than the growing cells by 1.7 ± 0.12 and 0.4 ± 0.1 log10 reduction, respectively, after 15 min of illumination. These data demonstrate that CHL excited with red light together with PEI is promising for the eradication of P. aeruginosa, and the susceptibility of P. aeruginosa to CHL/PEI is influenced by the concentrations and the exposure time.

Antibacterial and Antibiofilm Properties of the Alexidine Dihydrochloride (MMV396785) against Acinetobacter baumannii.

Antibiotic-resistant Acinetobacter baumannii infections among patients in hospital settings are rising at an alarming rate. The World Health Organization has designated carbapenem-resistant A. baumannii as a priority pathogen for drug discovery. Based on the open drug discovery approach, we screened 400 compounds provided as a Pandemic Response Box by MMV and DNDi to identify compounds with antibacterial and antibiofilm activity against two A. baumannii reference strains using a highly robust resazurin assay. In vitro screening identified thirty compounds with MIC ≤ 50μM having growth inhibitory properties against the planktonic state. Five compounds, with MMV IDs MMV396785, MMV1578568, MMV1578574, MMV1578564, and MMV1579850, were able to reduce metabolically active cells in the biofilm state. Of these five compounds, MMV396785 showed potential antibacterial and antibiofilm activity with MIC, MBIC, and MBEC of 3.125 μM, 12.5, and 25-100 µM against tested A. baumannii strains, respectively, showing biofilm formation inhibition by 93% and eradication of pre-formed biofilms by 60-77.4%. In addition, MMV396785 showed a drastic reduction in the surface area and thickness of biofilms. Further investigations at the molecular level by qRT-PCR revealed the downregulation of biofilm-associated genes when exposed to 50 µM MMV396785 in all tested strains. This study identified the novel compound MMV396785 as showing potential in vitro antibacterial and antibiofilm efficacy against A. baumannii.

Metabolomics investigation of global responses of Cronobacter sakazakii against common sanitizing in infant formula processing environments

Cronobacter sakazakii, an opportunistic bacterium, has raised a serious outbreak in powdered infant formula recent years. In this work, four sanitizing strategies used during infant formula processing, including chlorine, quaternary ammonium chloride (QAC), 60 °C heating, and malic acid (MA), were utilized against C. sakazakii among planktonic, air-dried (A), and air-dried & washed (AW) state, followed by an exploration of the metabolic responses induced by these treatments via a dual-platform metabolomics analysis with the ultra-high performance liquid chromatography-mass spectrometry and nuclear magnetic resonance. In the planktonic state, MA was the most effective in inhibiting bacterial growth, followed by chlorine, QAC, and 60 °C heating. Under A state, the efficacy of heating improved considerably, compared to that in the planktonic state, and remained unaltered under AW state. Chlorine and QAC were ineffective to control bacterial growth under A state, but their efficacy rose under AW state. Furthermore, the metabolomic analysis revealed chlorine induces amino acids catabolism, membrane lysis, and depression in carbohydrate and nucleotide metabolism in both planktonic and AW states, while the initiation of antioxidation mechanism was only found under AW state. Although the metabolic change caused by QAC in the planktonic state was similar to chlorine, the accumulation of osmoprotectant and membrane phospholipids within the AW cells reflected the effort to restore intracellular homeostasis upon QAC. Heating was characterized by considerable amino acid anabolism, along with mildly perturbed carbohydrate and nucleotide metabolism for heat shock protein preparation in both states. Lastly, MA promoted amino acid-dependent acid resistance under the planktonic state, and the regulation of antioxidation and osmoprotection under AW state. The metabolomics study elucidated the intracellular perturbation induced by common sanitizing, as well as the bacterial response, which provides insights for novel sanitization development.

Probiotics and novel probiotic delivery systems

Necrotizing enterocolitis (NEC) is an infectious and inflammatory intestinal disease that is the most common surgical emergency in the premature patient population. Although the etiology of the disease is multifactorial, intestinal dysbiosis is a hallmark of this disease. Based on this, probiotics may play a therapeutic role in NEC by introducing beneficial bacteria with immunomodulating, antimicrobial, and anti-inflammatory functions into the gastrointestinal tract. Currently, there is no Food and Drug Administration (FDA)-approved probiotic for the prevention and treatment of NEC. All probiotic clinical studies to date have administered the bacteria in their planktonic (free-living) state. This review will discuss established probiotic delivery systems including planktonic probiotics, prebiotics, and synbiotics, as well as novel probiotic delivery systems such as biofilm-based and designer probiotics. We will also shed light on whether or not probiotic efficacy is influenced by administration with breast milk. Finally, we will consider the challenges associated with developing an FDA-approved probiotic for NEC.

Identification of endometrial biofilm-specific proteins for in vivo diagnostics

Bacteria enter the uterus in a free-floating planktonic state, which makes them vulnerable to the host-immune system as well as exposure to effective antimicrobials. If not eliminated in a timely fashion, the bacteria will adhere to the endometrial surface and may form micro-colonies, develop a complex matrix, and switch into a biofilm community. The ability of bacteria to shift from planktonic to biofilm phenotypes has been described for both Gram-negative and Gram-positive bacteria in human mucosal tissue, indwelling catheters, and implants as well as other organs, including the equine endometrium. Unfortunately, there is currently no available assay to diagnose the condition in vivo. Literature in human medicine suggests that biofilm matrices contain both secretory and non-secretory proteins, and while some of the proteins are common among all biofilms, others may be specific for bacterial species. Therefore, the objective of this research was to identify specific proteins from biofilm-producing bacteria from mares with endometritis. Whole-cell extracts from potentially biofilm-forming bacterial isolates of Streptococcus zooepidemicus (SEZ) (n=3), Pseudomonas aeruginosa (n=1), and Escherichia coli (n=4) were analyzed by 2-D PAGE. Differentially expressed proteins were selected by overlapping 2-D PAGE biofilm proteins with planktonic culture proteins.In-gel analysis of differences (with a 3-fold cut-off)identified a total of 96 proteins for SEZ, 83 proteins for P. aeruginosa, and 69 proteins for E. coli as potential biofilm markers. Select proteins were further identified by MALDI-TOF/TOF. In SEZ, glutathione-disulfide reductase (48.8 kDa, PI 5.6), which catalyzes the reduction of glutathione disulfide to glutathione, a major cellular antioxidant, and TIGR00266 (25.3 kDa, PI 5.4) were identified as proteins of interest. For P. aeruginosa, thioredoxin A (11.8 kDa, PI 4.7), which participates in various redox reactions, osmotically inducible protein C (15.6 kDa, PI 5.9), important for cellular defense mechanism against oxidative stress, and bacterial extracellular solute-binding s, 3 family protein (33 kDa, PI 8.3) serve as potential targets. Potential biomarker candidates for E. coli include the serine protease pet autotransporter (139 kDa, PI 6.5) and outer membrane protein X (18 kDa, PI 6.6), a protein involved in numerous physiological processes including invasion, signal transduction, and binding external proteins. In conclusion, we identified biofilm-specific proteins that may serve as in vivo diagnostic markers for biofilms in uterine lavage from mares with biofilm-associated bacterial endometritis. Future research will pursue these proteins of interest for their use as diagnostic tools in the field.

Cinnamaldehyde reduces Yersinia enterocolitica activity and biofilm formation by altering membrane permeability and signalling

Yersinia enterocolitica is a foodborne pathogen that can infect food not only in its planktonic state but also as a biofilm on contact surfaces, making recurrent infections difficult to control. This article discusses various ways in which cinnamaldehyde can combat Y. enterocolitica. These include its effects on the permeability, integrity, and amino acid structure of cell membranes as well as on intracellular energy metabolism. We also investigated the effects of cinnamaldehyde on the formation of biofilms by Y. enterocolitica, including interference with quorum-sensing pathways, bacterial motility and adhesion, extracellular polymer production, and metabolic activity within the biofilm. Our results demonstrated that the minimum concentration of cinnamaldehyde required to effectively inhibit Y. enterocolitica is 0.625 mg/mL. It causes cell death by damaging the membrane, disrupting oxidative respiratory processes, interfering with cellular homeostasis, and disrupting cell function. Correspondingly, a concentration of 0.078 mg/mL cinnamaldehyde can suppress the production of biofilm and signalling molecules. This research aims to explore the mechanism by which cinnamaldehyde inhibits the activity of Y. enterocolitica and its biofilm, with the goal of providing more insight into whether cinnamaldehyde can be used as a natural, plant-based preservative and anti-biofilm agent.

High Disinfectant Tolerance in Pseudomonas spp. Biofilm Aids the Survival of Listeria monocytogenes.

Pseudomonas spp. are the most commonly found bacteria in food-processing environments due to properties such as a high growth rate at low temperatures, a high tolerance of antimicrobial agents, and biofilm formation. In this study, a set of Pseudomonas isolates originating from cleaned and disinfected surfaces in a salmon processing facility were screened for biofilm formation at 12 °C. A high variation in biofilm formation between the isolates was observed. Selected isolates, in both planktonic and biofilm states, were tested for resistance/tolerance to a commonly used disinfectant (peracetic acid-based) and antibiotic florfenicol. Most isolates showed a much higher tolerance in the biofilm state than in the planktonic state. In a multi-species biofilm experiment with five Pseudomonas strains with and without a Listeria monocytogenes strain, the Pseudomonas biofilm appeared to aid the survival of L. monocytogenes cells after disinfection, underscoring the importance of controlling the bacterial load in food-processing environments.

Comparison of Staphylococcus aureus tolerance between antimicrobial blue light, levofloxacin, and rifampin.

Bacterial biofilms readily develop on all medical implants, including percutaneous osseointegrated (OI) implants. With the growing rate of antibiotic resistance, exploring alternative options for managing biofilm-related infections is necessary. Antimicrobial blue light (aBL) is a unique therapy that can potentially manage biofilm-related infections at the skin-implant interface of OI implants. Antibiotics are known to have antimicrobial efficacy disparities between the planktonic and biofilm bacterial phenotypes, but it is unknown if this characteristic also pertains to aBL. In response, we developed experiments to explore this aspect of aBL therapy. We determined minimum bactericidal concentrations (MBCs) and antibiofilm efficacies for aBL, levofloxacin, and rifampin against Staphylococcus aureus ATCC 6538 planktonic and biofilm bacteria. Using student t-tests (p < 0.05), we compared the efficacy profiles between the planktonic and biofilm states for the three independent treatments and a levofloxacin + rifampin combination. Additionally, we compared antimicrobial efficacy patterns for levofloxacin and aBL against biofilms as dosages increased. aBL had the most significant efficacy disparity between the planktonic and biofilm phenotypes (a 2.5 log10 unit difference). However, further testing against biofilms revealed that aBL had a positive correlation between increasing efficacy and exposure time, while levofloxacin encountered a plateau. While aBL efficacy was affected the most by the biofilm phenotype, its antimicrobial efficacy did not reach a maximum. We determined that phenotype is an important characteristic to consider when determining aBL parameters for treating OI implant infections. Future research would benefit from expanding these findings against clinical S. aureus isolates and other bacterial strains, as well as the safety of long aBL exposures on human cells.