Log Colony-forming Units Research Articles

The Effect of Commonly Used Organic Acids on the Microbiota of Cured Deli-Style Turkey Breast

The purpose of this study was to investigate changes in the microbial community structure consisting of spoilage lactic acid bacteria (LAB) when sodium lactate (SL) and SL + sodium diacetate (SD) are included in RTE meat product formulations at concentrations commonly used for controlling Listeria monocytogenes. Sliced cooked, vacuum-packaged turkey breast samples containing no SD or SL (control [C]), 0.125% SD, and the combination of 2.5% SL + 0.125%SD (SLSD) were inoculated with a target of 3 log colony-forming units (CFU)/g of 5 different LAB species (Lactobacillus sakei, Leuconostoc mesenteroides [bacon and deli-shaved ham isolates], Lactococcus lactis, and Enterococcus faecium) and stored at 4°C for 35 d. Microbial community changes were analyzed utilizing 16S ribosomal RNA (rRNA) gene sequencing for the V4 region from the samples collected at days 0, 7, 14, 21, and 35. No significant difference (P>0.05) was observed between the richness of microbial community for all treatments. According to Bray-Curtis dissimilarity matrix and permutational analysis of variance, a significant difference in β-diversity was observed only between C and SLSD (P<0.05) because of the antimicrobial effect of the addition of SL that slowed down changes in microbial community composition until day 14. L. sakei remained a dominant strain throughout 35 d of storage regardless of treatment, whereas L. mesenteroides existed with a very low abundance. Two of the five strains were not seen after day 7. No significant effect (P>0.05) was observed for the SD treatment compared with C in the microbiota. According to the results from this study, only SLSD affected the microbial community structure at the beginning of the storage. This study demonstrated that the incorporation of SL in the formulation slowed down the microbial spoilage and the changes in the microbiota of RTE meat products.

A novel antibacterial compound decreases MRSA biofilm formation without the use of antibiotics in a murine model.

Despite significant advancements in material science, surgical site infection (SSI) rates remain high and prevention is key. This study aimed to demonstrate the in vivo safety and antibacterial efficacy of titanium implants treated with a novel broad-spectrum biocidal compound (DBG21) against methicillin-resistant Staphylococcus aureus (MRSA). Titanium (Ti) discs were covalently bound with DBG21. Untreated Ti discs were used as controls. All discs were implanted either untreated for 44 control mice or DBG21-treated for 44 treated mice. After implantation, 1 × 107 colony forming units (CFU) of MRSA were injected into the operating site. Mice were killed at 7 and 14 daysto determine the number of adherent bacteria (biofilm) on implants and in the peri-implant surrounding tissues. Systemic and local toxicity were assessed. At both 7 and 14 days, DBG21-treated implants yielded a significant decrease in MRSA biofilm (3.6 median log10 CFU [99.97%] reduction [p < 0.001] and 1.9 median log10 CFU [98.7%] reduction [p = 0.037], respectively) and peri-implant surrounding tissues (2.7 median log10 CFU/g [99.8%] reduction [p < 0.001] and 5.6 median log10 CFU/g [99.9997%] reduction [p < 0.001], respectively). There were no significant differences between control and treated mice in terms of systemic and local toxicity. DBG-21 demonstrated a significant decrease in the number of biofilm bacteria without associated toxicity in a small animal implant model of SSI. Preventing biofilm formation has been recognized as a key element of preventing implant-related infections.

Investigating the Ability of Edwardsiella ictaluri and Flavobacterium covae to Persist within Commercial Catfish Pond Sediments under Laboratory Conditions.

Two prevalent bacterial diseases in catfish aquaculture are enteric septicemia of catfish and columnaris disease caused by Edwardsiella ictaluri and Flavobacterium covae, respectively. Chronic and recurring outbreaks of these bacterial pathogens result in significant economic losses for producers annually. Determining if these pathogens can persist within sediments of commercial ponds is paramount. Experimental persistence trials (PT) were conducted to evaluate the persistence of E. ictaluri and F. covae in pond sediments. Twelve test chambers containing 120 g of sterilized sediment from four commercial catfish ponds were inoculated with either E. ictaluri (S97-773) or F. covae (ALG-00-530) and filled with 8 L of disinfected water. At 1, 2, 4-, 6-, 8-, and 15-days post-inoculation, 1 g of sediment was removed, and colony-forming units (CFU) were enumerated on selective media using 6 × 6 drop plate methods. E. ictaluri population peaked on Day 3 at 6.4 ± 0.5 log10 CFU g-1. Correlation analysis revealed no correlation between the sediment physicochemical parameters and E. ictaluri log10 CFU g-1. However, no viable F. covae colonies were recovered after two PT attempts. Future studies to improve understanding of E. ictaluri pathogenesis and persistence, and potential F. covae persistence in pond bottom sediments are needed.

A do-it-yourself test protocol using commercial Bacillus atrophaeus spores to evaluate the effectiveness of ultraviolet-C light room-decontamination devices.

We developed a do-it-yourself test protocol using commercial Bacillus atrophaeus spores to assess the efficacy of ultraviolet-C (UV-C) light room-decontamination devices. Overall, 4 UV-C devices reduced B. atrophaeus by ≥3 log10 colony-forming units in 10 minutes, whereas a smaller device required 60 minutes. Of 10 in-use devices, only 1 was ineffective.

Comparative Evaluation of Antimicrobial Activity of Herbal and Nonherbal Root Canal Irrigants on Enterococcus faecalis - An In vitro Study

Introduction: One of the most common pathogens in infected root canals is Enterococcus faecalis. Even though, the role of irrigants in controlling pathogens is well established, there is a chance for drug resistance and cytotoxicity in using chemical irrigants, Therefore newer researches have forced the man to look the herbal way. Aim of the Study: To assess and the antimicrobial effects of nonherbal and herbal root canal irrigants on E. faecalis. Methodology: The study was conducted on 110 permanent extracted human teeth. Culture suspension of E. faecalis was inoculated in each root canal and were divided randomly into groups, (i): Herbal group [QMixTM 2 in 1, Endoseptone, Biopure mixture of tetracycline, acid and detergent (MTAD)] and (ii) Nonherbal group (Morinda citrifolia juice, Triphala juice, Coconut milk), and control group (Sodium hypochlorite and Distilled water). Colony forming units (CFUs) were evaluated and counted at baseline (S0), immediately after irrigation (S1) and after 3 days (S2). The obtained mean of log CFU was compared using Friedman’s two-way ANOVA. Results: There was a significant reduction in CFU counts in both herbal and nonherbal group. The greatest antimicrobial effects were observed in nonherbal group against E. faecalis. Conclusion: The overall antimicrobial effects of different irrigants were maximum for the nonherbal group, whereas the herbal group also showed significant reductions. The herbal irrigants hold the promise of becoming efficient irrigants and warrant further research.

Solar-driven efficient heterogeneous subminute water disinfection nanosystem assembled with fingerprint MoS2

Although heterogeneous water disinfection can avoid secondary pollution and other shortcomings in homogeneous systems, its low disinfection efficiency seriously hinders its development. Here we successfully address the aforementioned issues of heterogeneous disinfection by developing discrete nanoflakes of (Al2O3@v-MoS2)/Cu/Fe3O4. Three exciting features are integrated into such a novel structure: bifacial vertically aligned nanofingerprint MoS2 grown on both sides of the light-transparent Al2O3 nanoflakes that can largely absorb sunlight, where both sides can operate simultaneously; a Cu-MoS2 junction that enhances charge separation for the efficient generation of reactive oxygen species; and magnetic Fe3O4 nanoparticles that have magnetic separation capability and conveniently regenerate after disinfection. The (Al2O3@v-MoS2)/Cu/Fe3O4 nanostructures reported herein exhibit outstanding water disinfection with thorough inactivation of over 5.7 log10 colony-forming units ml−1 Escherichia coli within 1 min in real sunlight (the system thermal effect has little impact on disinfection performances) as well as facile separation and stable long cycle reuse, demonstrating broad application prospects. Heterogeneous water disinfection is a promising way to avoid secondary pollution, but it is not very efficient. The development of nanoflakes shows that a much higher efficiency than previously achieved can be achieved through solar-driven heterogeneous disinfection.

Application of MALDI-TOF MS for enumerating bacterial constituents of defined consortia

Characterization of live biotherapeutic product (LBP) batches typically includes a measurement of viability, such as colony forming units (CFU). However, strain-specific CFU enumeration assays can be complicated by the presence of multiple organisms in a single product with similar growth requirements. To overcome specific challenges associated with obtaining strain-specific CFU values from multi-strain mixtures, we developed a method combining mass spectrometry-based colony identification with a traditional CFU assay. This method was assessed using defined consortia made from up to eight bacterial strains. Among four replicate batches of an eight-strain mixture, observed values differed from expected values by less than 0.4 log10 CFU among all strains measured (range of differences, -0.318 to + 0.267). The average difference between observed and expected values was + 0.0308 log10 CFU, with 95% limits of agreement from -0.347 to 0.408 (Bland–Altman analysis). To estimate precision, a single batch of eight-strain mixture was assayed in triplicate by three different users, for a total of nine measurements. Pooled standard deviation values ranged from 0.067 to 0.195 log10 CFU for the eight strains measured, and user averages did not differ significantly. Leveraging emerging mass-spectrometry-based colony identification tools, a novel method for simultaneous enumeration and identification of viable bacteria from mixed-strain consortia was developed and tested. This study demonstrates the potential for this approach to generate accurate and consistent measurements of up to eight bacterial strains simultaneously and may provide a flexible platform for future refinements and modifications.Key points• Enumeration of live biotherapeutics is essential for product quality and safety.• Conventional CFU counting may not differentiate between strains in microbial products.• This approach was developed for direct enumeration of mixed bacterial strains simultaneously.

Antimicrobial efficacy of cyclic α- and β-peptides incorporated in polyurethane coatings.

Microbial growth on surfaces poses health concerns and can accelerate the biodegradation of engineered materials and coatings. Cyclic peptides are promising agents to combat biofouling because they are more resistant to enzymatic degradation than their linear counterparts. They can also be designed to interact with extracellular targets and intracellular targets and/or self-assemble into transmembrane pores. Here, we determine the antimicrobial efficacy of two pore-forming cyclic peptides, α-K3W3 and β-K3W3, against bacterial and fungal liquid cultures and their capacity to inhibit biofilm formation on coated surfaces. These peptides display identical sequences, but the additional methylene group in the peptide backbone of β-amino acids results in a larger diameter and an enhancement in the dipole moment. In liquid cultures, β-K3W3 exhibited lower minimum inhibitory concentration values and greater microbicidal power in reducing the number of colony forming units (CFUs) when exposed to a gram-positive bacterium, Staphylococcus aureus, and two fungal strains, Naganishia albida and Papiliotrema laurentii. To evaluate the efficacy against the formation of fungal biofilms on painted surfaces, cyclic peptides were incorporated into polyester-based thermoplastic polyurethane. The formation of N. albida and P. laurentii microcolonies (105 per inoculation) for cells extracted from coatings containing either peptide could not be detected after a 7-day exposure. Moreover, very few CFUs (∼5) formed after 35 days of repeated depositions of freshly cultured P. laurentii every 7 days. In contrast, the number of CFUs for cells extracted from the coating without cyclic peptides was >8 log CFU.

Survival of Candida auris on environmental surface materials and low-level resistance to disinfectant

Candida auris persists in the environment despite disinfection. Its survival on various environmental surfaces and the effect of sublethal concentrations of disinfectants on C.auris has not been studied. To investigate the survival of C.auris on environmental surfaces, and the effect of sublethal concentrations of disinfectants. Surface material blocks were fabricated and artificially contaminated with C.auris. Viable counts were assessed for 3 weeks. In addition, C.auris cells were pulsed daily with disinfectants for 15 days, and minimum inhibitory concentrations (MICs) were determined. Ergosterol quantities and efflux pump assays were performed on disinfectant-exposed strains using standard methods. C.auris survived on all the surfaces for >3 weeks, with the lowest count of 2.3 log colony-forming units, regardless of wet or dry conditions. Wet wood supported the growth of C.auris (a 1 log increase), whereas dry wood inhibited this organism (both P<0.01). In the biofilm form, C.auris flourished on all surfaces. Although the MICs increased in C.auris cells pulsed with sodium dichloroisocyanurate and benzalkonium chloride, only C.auris exposed to benzalkonium chloride showed decreased ergosterol content and an activated efflux pump. Although C.auris survived on all tested surfaces, survival on wet wood was remarkable. C.auris pulsed with benzalkonium chloride developed some degree of tolerance to disinfectant and showed efflux pump activation, suggesting the development of low-level resistance.

Differences in Decreasing Bacteria Colonization Wound Irrigation using 0.9% Nacl Compared with Wound Irrigation using Active Ingredients Polyhexanide 0.1% and 0.1% Betaine in Open Fractures of Long Bone

BACKGROUND: An open fracture is an emergency case that must be treated quickly to prevent infection. The use of an irrigation solution containing 0.1% of the antimicrobial polyhexanide and 0.1% of the surfactant betaine has been associated with more satisfactory wound healing. AIM: This study was comparing irrigation with polyhexanide 0.1% and betaine 0.1% (P + B) in reducing bacterial colonization of open fractures compared to using 0.9% natrium chloride (NaCl) solution. METHODS: This study uses a pre- and post-control group design. Sampling was carried out in the operation room of the emergency care installation, inpatient installation, and outpatient installation of prof. Hospital. Dr. I.G.N.G Ngoerah Denpasar, between September and November 2022. From a sample population suffering from open fractures of the lower extremities, samples of patients were taken who were irrigated with 0.9% NaCl solution with a mixture of polyhexanide 0.1% and betaine 0.1%. The presence or absence of bacterial colonization was examined using a surgical wound base swab with the Levine technique. RESULTS: In the 30 patients in this study, there was a significant decrease in bacterial levels (Log colony-forming unit [CFU]/g) after irrigation, both from irrigation with NaCl (p = 0.04) and NaCl + P + B (p &lt; 0.001). The reduction in bacterial levels was more significant with NaCl + P + B compared with NaCl alone, at post-irrigation (p &lt; 0.001), H+3 (p = 0.003), and H + 7 (p &lt; 0.001). CONCLUSION: Wound irrigation using a combination of 0.9% NaCl, 0.1% polyhexanide, and 0.1% betaine reduced bacterial colonization more than using 0.9% NaCl-only solution in open fractures of long bones.

Longitudinal Study for the Detection and Quantification of Campylobacter spp. in Dairy Cows during Milking and in the Dairy Farm Environment.

Campylobacteriosis outbreaks have repeatedly been associated with the consumption of raw milk. This study aimed to explore the variation in the prevalence and concentration of Campylobacter spp. in cows' milk and feces, the farm environment and on the teat skin over an entire year at a small German dairy farm. Bi-weekly samples were collected from the environment (boot socks), teats, raw milk, milk filters, milking clusters and feces collected from the recta of dairy cows. Samples were analyzed for Campylobacter spp., E. coli, the total aerobic plate count and for Pseudomonas spp. The prevalence of Campylobacter spp. was found to be the highest in feces (77.1%), completely absent in milking equipment and low in raw milk (0.4%). The mean concentration of Campylobacter spp. was 2.43 log10 colony-forming units (CFU)/g in feces and 1.26 log10 CFU/teat swab. Only a single milk filter at the end of the milk pipeline and one individual cow's raw milk sample were positive on the same day, with a concentration of 2.74 log10 CFU/filter and 2.37 log10 CFU/mL for the raw milk. On the same day, nine teat swab samples tested positive for Campylobacter spp. This study highlights the persistence of Campylobacter spp. for at least one year in the intestine of individual cows and within the general farm environment and demonstrates that fecal cross-contamination of the teats can occur even when the contamination of raw milk is a rare event.

Influence of different lactic acid bacteria strains and milling process on the solid-state fermented green and red lentils (Lens culinaris L.) properties including gamma-aminobutyric acid formation.

The aim of this study was to evaluate the influence of lactic acid bacteria (LAB) strains (Lactiplantibacillus plantarum No.122 and Lacticaseibacillus casei No.210) and milling process on the solid-state fermented (for 24 h, at 30°C) green and red lentils (Lens culinaris L.) properties, chiefly pH, LAB viable counts, color coordinates, free amino acid (FAA) profile, γ-aminobutyric acid (GABA) and biogenic amine (BA) concentrations, fatty acid (FA) and volatile compound (VC) profiles. Results showed that both of the tested LAB strains are suitable for the fermentation of lentils: pH of fermented lentils was <4.5 and LAB viable counts >8.0 log10 colony-forming units (CFU)/g. A very strong negative correlation was found (r = -0.973, p ≤ 0.0001) between LAB counts and pH of the samples. Also, fermentation and milling process were significant factors toward color coordinates of the lentils. In most of the cases, solid-state fermentation (SSF) increased essential FAA content in lentils; however, some of the non-essential FAA content was reduced. SSF significantly increased GABA concentration in lentils and milling process was a significant factor on GABA content of the samples (p ≤ 0.05). The main BA in lentils was spermidine, and SSF decreased their total BA content (34.8% on average in red lentils and 39.9% on average in green lentils). The main FA in lentils were linoleic and oleic. The main VC in lentils were hexanal, 1-hexanol, hexanoic acid, D-limonene and (E)-2-nonen-1-ol. Furthermore, most of the VC showed significant correlations with pH of lentil samples, LAB counts and FA content. Finally, the LAB strain used for fermentation and the milling process of lentils are significant factors for most of the analyzed parameters in lentil. Moreover, despite the higher GABA concentration found in green non-milled SSF lentils, application of combined milling and SSF is recommended because they showed the lowest BA content in addition to higher essential FAA and GABA concentrations.

Inhibitory Effect of Two Closely Related Phages on Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a foodborne pathogenic bacterium commonly found in seafood. The emergence of drug-resistant strains poses a threat to human public health and economic development. Therefore, there are increasing needs to develop new technologies in controlling multidrug-resistant V. parahaemolyticus strains and to evaluate their practical efficiency in seafood or mariculture. In this study, we screened two genetically related V. parahaemolyticus phages, F23s2 and H256D1, which belonged to the siphoviridae family and podoviridae family, respectively. They showed 97.13% and 96.13% identity with Vibrio phage vB_Vpap_MGD1, respectively. Both phages were stable at pH 4-11 and displayed temperature tolerance (<70°C). Meanwhile they showed a broad host spectrum for multidrug-resistant V. parahaemolyticus, and Phage F23s2 lysed 16 of all 23 V. parahaemolyticus strains, while phage H256D1 lysed 10 strains. Phage F23s2 and H256D1 had a good inhibitory effect on V. parahaemolyticus in shrimp meat. Compared with the negative group, the bacterial amount of experimental group with phage F23s2 decreased by 1.60 log colony-forming unit (CFU)/mL at 12 h. For phage H256D1, the bacterial concentration of shrimp meat contaminated with V. parahaemolyticus H256 increased to 5.65 log CFU/mL at 72 h, while the concentration of the experimental group in presence of phage H256D1 was 3.58 log CFU/mL. All live clams infected with V. parahaemolyticus died after 96 h in the absence of phage, whereas clams with phage F23s2 and H256D1 still had a survival rate of 12% and 4%, respectively. Understanding the gene function and biology of phages facilitates its application for control of V. parahaemolyticus contamination worldwide.

Isolation and characterization of a broad-spectrum phage SapYZU11 and its potential application for biological control of Staphylococcus aureus

Staphylococcus aureus, a prominent pathogen, is frequently encountered in clinical and food-processing settings. Given its ability to develop antimicrobial resistance, effective control strategies are required to ensure microbial safety. In this study, four lytic S. aureus phages (SapYZU10, SapYZU11, SapYZU12, and SapYZU13) were isolated from sewage samples in Yangzhou, China. Their biological characteristics and bactericidal effect against S. aureus isolates in vitro and in milk and fresh pork were evaluated. Their activities remained relatively stable under stressful conditions (-80–70°C, pH 3.0–12.0). Notably, SapYZU11 (100%, 53/53) effectively lysed all 53 S. aureus strains, followed by SapYZU12 (90.57%, 48/53), SapYZU13 (79.25%, 42/53), and SapYZU10 (71.70%, 38/53). Among the phages with short latent periods (10–20 min), SapYZU11 had a larger burst size (152.00 plaque forming units [PFU]/mL) and no genes related to antibiotic resistance and virulence. Furthermore, SapYZU11 effectively eradicated S. aureus and its cocktail (YZUsa1, YZUsa4, YZUsa12, YZUsa14, and methicillin-resistance S. aureus (MRSA) JCSC 4744) in Luria–Bertani broth and both food items. Particularly in milk, SapYZU11 with a multiplicity of infection (MOI) of 100 inhibited MRSA JCSC 4744 strain and S. aureus cocktail with maximum reduction levels of 5.03 log (Lg) colony-forming unit (CFU)/mL and 2.80 Lg CFU/mL, respectively. Conversely, contaminated pork treated with three MOIs of SapYZU11 at 25°C and 4°C resulted in reductions of 0.29–1.29 Lg CFU/mL and 0.11–0.32 Lg CFU/mL, respectively. Therefore, SapYZU11 proved as a promising biocontrol agent against S. aureus in different food production settings.

Evaluation of the antibacterial activity of gallic acid anchored phthalocyanine-doped silica nanoparticles towards Escherichia coli and Staphylococcus aureus biofilms and planktonic cells

In this work, we have described the synthesis of phthalocyanine complexes Zn(II) tetrakis 4-(5-formylpyridin-2-yl)oxy) phthalocyanine (2), Zn(II) tetrakis-1‑butyl‑4-(2-(6- (tetra-phenoxy)pyridin-3-yl) vinyl)pyridin-1-ium phthalocyanine (3) and Zn(II) tetrakis 1‑butyl‑5-(2-(1-butylpyridin-1-ium-4-yl)vinyl)-2-(tetra-phenoxy)pyridin-1-ium phthalocyanine (4). The effect of a varying number of charges when the Pc complexes are alone or grafted in gallic acid (GA) tagged silica nanoparticles on photodynamic antimicrobial chemotherapy (PACT) is investigated toward Staphylococcus aureus (S.aureus) and Escherichia coli (E.coli) in both planktonic and biofilm forms. Complex 4, bearing a total of 8 cationic charges, displayed the highest activity with log CFU (colony forming units) values of 8.60 and 6.42 against E.coli and S.aureus biofilms, respectively. The surface stability of E.coli and S.aureus biofilms in the presence of 4 and its conjugate was analyzed using cyclic voltammetry. Scanning electron microscopy (SEM) and Raman spectra are also used to study the conformational and biochemical changes within biofilm upon subjecting them to PACT.

Antimicrobial blue light as a biofilm management therapy at the skin-implant interface in an ex vivo percutaneous osseointegrated implant model.

Biofilm contamination is often present at the skin-implant interface of transfemoral osseointegrated implants leading to frequent infection, irritation, and discomfort. New biofilm management regimens are needed as the current standard of washing the site with soap and water is inadequate to manage infection rates. We investigated the potential of antimicrobial blue light, which has reduced risk of resistance development and broad antimicrobial mechanisms. Our lab developed an antimicrobial blue light(aBL) device uniquely designed for an ex vivo system based on an established ovine osseointegrated (OI) implant model with Staphylococcus aureus ATCC 6538 biofilms as initial inocula. Samples were irradiated with aBL or washed for three consecutive days after which they were quantified. Colony-forming unit (CFU) counts were compared with a control group (bacterial inocula without treatment). After 1 day, aBL administered as a single 6 h dose or two 1 h doses spaced 6 h apart both reduced the CFU count by 1.63 log10 ± 0.02 CFU. Over 3 days of treatment, a positive aBL trend was observed with a maximum reduction of ~2.7 log10 CFU following 6 h of treatment, indicating a relation between multiple days of irradiation and greater CFU reductions. aBL was more effective at reducing the biofilm burden at the skin-implant interface compared with the wash group, demonstrating the potential of aBL as a biofilm management option.

Anti-biofilm and bystander effects of antimicrobial photo-sonodynamic therapy against polymicrobial periopathogenic biofilms formed on coated orthodontic mini-screws with zinc oxide nanoparticles.

The present study evaluated the anti-biofilm and bystander effects of antimicrobial photo-sonodynamic therapy (aPSDT) on the polymicrobial periopathogenic biofilms formed on mini-screws coated with zinc oxide nanoparticles (ZnONPs). Thirty orthodontic identical mini-screws were divided into 6 groups (n=5) as follows: 1. negative control: uncoated mini-screw+phosphate-buffered saline (PBS), 2. positive control: uncoated mini-screw+0.2% CHX, 3. coating control: coated mini-screw+PBS, 4. antimicrobial photodynamic therapy (aPDT): coated mini-screw+light emitting diode (LED), 5. Antimicrobial sonodynamic therapy (aSDT): coated mini-screw+ultrasound waves, and 6. aPSDT: coated mini-screw+LED+ultrasound waves. Electrostatic spray-assisted vapor deposition was employed to coat ZnONPs on titanium mini-screws. The biofilm inhibition test was used to assess the anti-biofilm efficacy against polymicrobial periopathogenic biofilms including Porphyromonas gingivitis, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans, and the results were shown as the percent reduction of Log10 colony-forming unit (CFU)/mL. Following each treatment, the gene expression levels of TNF-α, IL-1β, and IL-6 were evaluated on human gingival fibroblast (HGF) cells viaquantitative real-time polymerase chain reaction (qRT-PCR) to reveal the bystander effects of aPSDT on HGF cells. A significant reduction in log10 CFU/mL of periopathogens was observed in groups treated with aPDT, aSDT, aPSDT, and 0.2% CHX up to 6.81, 6.63, 5.02, and 4.83 log, respectively, when compared with control groups (P<0.05). 0.2% CHX and aPSDT groups demonstrated significantly higher capacity in eliminating the periopathogen biofilm compared with other groups (P<0.05). The qRT-PCR showed that the expression level of inflammatory cytokines was significantly down regulated in aPDT, aSDT, and aPSDT groups (P<0.05). It was found that the ZnONPs-mediated aPSDT could significantly reduce periopathogen biofilm as well as the expression level of inflammatory cytokines.

Ex vivo wound model on porcine skin for the evaluation of the antibiofilm activity of polyhexamethylene biguanide and ciprofloxacin.

This study aimed to standardize the use of an ex vivo wound model for the evaluation of compounds with antibiofilm activity. The in vitro susceptibility of Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853 to ciprofloxacin and polyhexamethylene biguanide (PHMB) was evaluated in planktonic and biofilm growth. The effects of ciprofloxacin and PHMB on biofilms grown on porcine skin explants were evaluated by colony-forming unit (CFU) counting and confocal microscopy. Minimum inhibitory concentrations (MICs) against S. aureus and P. aeruginosa were, respectively, 0.5 and 0.25 µg mL-1 for ciprofloxacin, and 0.78 and 6.25 µg mL-1 for PHMB. Minimum biofilm eradication concentrations (MBECs) against S. aureus and P. aeruginosa were, respectively, 2 and 8 µg mL-1 for ciprofloxacin, and 12.5 and>25 µg mL-1 for PHMB. Ciprofloxacin reduced (P<0.05) log CFU counts of the biofilms grown ex vivo by 3 and 0.96 for S. aureus and P. aeruginosa, respectively, at MBEC, and by 0.58 and 8.12 against S. aureus and P. aeruginosa, respectively, at 2xMBEC. PHMB (100 µg/mL) reduced (P<0.05) log CFU counts by 0.52 for S. aureus and 0.68 log for P. aeruginosa, leading to an overall decrease (P<0.05) in biofilm biomass. The proposed methodology to evaluate the susceptibility of biofilms grown ex vivo led to reproducible and reliable results.

Encapsulation of Lactobacillus plantarum ELB90 by electrospraying in a double emulsion (W1/O/W2) loaded alginate beads to improve the gastrointestinal survival and thermal stability.

In the present study, the Lactobacillus plantarum ELB90 was encapsulated in double emulsion (W1/O/W2) loaded alginate beads (emulbeads) by electrospraying and compared with emulsion-free control beads. The viability of encapsulated and free cells was assessed by exposing them to thermal processing (65 °C for 30 min and 72 °C for 3min) and simulated gastrointestinal conditions. The beads were characterized by optical, scanning electron, fluorescence, and confocal laser scanning microscopy, as well as Fourier transform infrared and gel strength analysis. After the intestinal stage of digestion, the survival rate of free bacteria was 38% [3.70 log colony-forming units (CFU) g-1 ], only increased to 43% and 53% for bare and chitosan-coated control beads, and it elevated the survival rate to 75% and 84% (8.70 log CFU g-1 ) for bare and chitosan-coated emulbeads, respectively. The presence of inulin increased gastrointestinal viability only in uncoated emulbeads. The bacteria loaded in emulbeads retained greater viability (5.90-6.90 log CFU g-1 ) against thermal treatment, compared to control beads (2.07-4.10 log CFU g-1 ) and free bacteria (2.57-3.11 log CFU mL-1 ). Encapsulation of L. plantarum ELB90 only in emulsion-free beads may not be appropriate for providing thermal stability. Inulin addition and chitosan-coating of the beads increased the size, and emulbeads presented smoother surfaces compared to emulsion-free beads. The contribution of a double emulsion into the gel matrix of electrosprayed alginate beads exhibited enhanced protection for probiotic bacteria that could be useful for the development of functional foods. © 2023 Society of Chemical Industry.

Control of Mycobacterium avium subsp. paratuberculosis load within infected bovine monocyte-derived macrophages is associated with host genetics.

The genetic loci influencing individual resistance to Mycobacterium avium subsp. paratuberculosis (MAP) infection are still largely unknown. In the current study, we searched for genetic loci associated with resistance to MAP infection by evaluating the performance of monocyte-derived macrophages (MDMs) isolated from the peripheral blood of 75 healthy Holsteins cows and infected ex vivo with MAP. Bacterial load (log colony-forming units, log CFUs) within MDMs was quantified at 2h and 7 days p. i. using a BACTEC MGIT 960 instrument. In addition, the expression levels of some genes with important roles in the innate immune response including epiregulin (EREG), complement component C3 (C3), galectin-9 (Gal9), and nitric oxide (NO-) were measured in the supernatant of the infected cells. DNA from peripheral blood samples of the animals included in the study was isolated and genotyped with the EuroG MD bead Chip (44,779 single nucleotide-polymorphisms, SNPs). Linear mixed models were used to calculate the heritability (h2 ) estimates for each indicator of MDM performance, MAP load within MDMs and EREG, C3, Gal9, and NO-expression. After performing a genome-wide association study, the only phenotypes that showed SNPs with a significant association were the bacterial load within MDMs at 2h (h2 = 0. 87) and 7 days (h2 = 0.83) p.i. A total of 6 SNPs, 5 candidate genes, and one microRNA on the Bos taurus chromosomes BTA2, BTA17, BTA18, and BTA21 were associated with MAP load at 2h p.i. Overlap was seen in two SNPs associated with the log CFUs at 2h and 7d p.i. The identified SNPs had negative regression coefficients, and were, therefore, associated with a low bacterial load within MDMs. Some of the identified SNPs were located within QTLs previously associated with longevity, reproductive, and udder health traits. Some of the identified candidate genes; Oxysterol Binding Protein Like 6, Cysteine and Serine Rich Nuclear Protein 3, and the Coiled-Coil Domain Containing 92 regulate cellular cholesterol trafficking and efflux, apoptosis, and interferon production, respectively. Taken together, our results define a heritable and distinct immunogenetic profile in MAP-infected macrophages designed to limit bacterial load early after infection.