Colony-forming Unit Counts Research Articles

Decontaminative Properties of Cold Atmospheric Plasma Treatment on Collagen Membranes Used for Guided Bone Regeneration.

Background cold atmospheric plasma (CAP) is known to be a surface-friendly yet antimicrobial and activating process for surfaces such as titanium. The aim of the present study was to describe the decontaminating effects of CAP on contaminated collagen membranes and their influence on the properties of this biomaterial in vitro. Material and Methods: A total of n = 18 Bio-Gide® (Geistlich Biomaterials, Baden-Baden, Germany) membranes were examined. The intervention group was divided as follows: n = 6 membranes were treated for one minute, and n = 6 membranes were treated for five minutes with CAP using kINPen® MED (neoplas tools GmbH, Greifswald, Germany) with an output of 5 W, respectively. A non-CAP-treated group (n = 6) served as the control. The topographic alterations were evaluated via X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). Afterward, the samples were contaminated with E. faecalis for 6 days, and colony-forming unit (CFU) counts and additional SEM analyses were performed. The CFUs increased with CAP treatment time in our analyses, but SEM showed that the surface of the membranes was essentially free from bacteria. However, the deeper layers showed remaining microbial conglomerates. Furthermore, we showed, via XPS analysis, that increasing the CAP time significantly enhances the carbon (carbonyl group) concentration, which also correlates negatively with the decontaminating effects of CAP. Conclusions: Reactive carbonyl groups offer a potential mechanism for inhibiting the growth of E. faecalis on collagen membranes after cold atmospheric plasma treatment.

Live, Probiotic, or Neither? Microbial Composition of Retail-Available Kombucha and “Hard” Kombucha in the Pacific Northwest of the United States

Kombucha is an acidic non-alcoholic fermented tea beverage that has surged in popularity over the past decade. A key driver of this popularity is the perception that kombucha contains microbes that contribute to gut health, a perception that is reinforced by package claims such as “live” and “probiotic”. We surveyed retail-available non-alcoholic (soft) and alcoholic (hard) kombucha and selected 39 products, of which 74.4% included at least one of these claims. Based upon Nuclear Magnetic Resonance (NMR) evaluation of the chemical composition, approximately one-third of soft kombucha products were re-categorized as “soft-aberrant” on the basis of their alcohol by volume (ABV) exceeding 0.5%. Across all three categories, the majority (82.1%) of products were “live”, containing culturable yeast and/or bacteria. However, there were no significant differences in median maximum colony-forming unit (CFU) counts between these categories, nor was there a difference according to package claims. Interestingly, only 6.3% of soft and 10% of soft-aberrant kombucha products exceeded 106 CFU/mL, the threshold that would deliver at least one billion cells in one package. None of the sampled hard kombucha exceeded this threshold. Metabarcoding analyses of microbial communities revealed differences in composition between categories and according to package claims. Notably, the subset of products with “probiotic” claims that listed Bacillus coagulans as an ingredient were enriched in read counts for Bacillus.

Source strengths obtained with three clean air suits fulfilling the requirements for high performance in EN 13795-2:2019: An experimental study

Introduction Clothing made from tight material worn by operating room staff adds to the effect achieved by ventilation in the effort to keep a low level of bacteria in the operating room air. The material used for clean air suits should be tested according to the standard EN 13795-2. Aim The aim of the study was to investigate whether there was a difference in protective capacity between three clean air suits made from materials fulfilling the requirements of EN 13795-2:2019 and designed as described in Annex E of the standard. Materials and methods Eight people (five men and three women) performed standardized movements in a dispersal chamber with a fixed supply air flow. Each person performed two test cycles with each clean air suit. Counts of colony-forming units (CFU) in the air were measured during testing and source strength was calculated for each test cycle. Results The mean values of source strength for the three clean air suits were 1.4 CFU/s, 0.8 CFU/s and 0.7 CFU/s. The difference between the most and the least protective garment was statistically significant (p < 0.05). Conclusions Clean air suits made from material fulfilling the requirements for ‘high performance’ in the standard EN 13795-2:2019 might show a significant difference in protective capacity when comparing source strength. Tests for measuring of source strength in a dispersal chamber can be performed as suggested in Annex E of the standard, i.e. ‘The test person is male, 20–50 years old, with no visible skin disorder’.

Endophytic Strains of <i>Bacillus thuringiensis</i> for the Development of Means to Control the Number of the Colorado Potato Beetle in Potato Crops

Currently, the search for B. thuringiensis strains to be used as biological control agents is based on their toxicity to insects in laboratory tests. At the same time, a number of strains of these bacteria are able to exist in symbiotic relationships with host plants, including being endophytes. The ability of B. thuringiensis strains to penetrate into the internal tissues of plants was evaluated by counting colony-forming units (CFU) of microorganisms 7 days after inoculation of sterile potato plants in test tubes; insecticidal activity was tested on larvae of the 3rd age obtained from overwintered adults of the Colorado beetle collected from fields in the Chishminsky and Iglinsky districts of Bashkortostan. It was shown that the strain of B. thuringiensis B-5351, which inhabits the surface (50.01 ± 8.10 CFU × 105/g) and internal tissues (38.92 ± 9.62 CFU × 105/g) of plant shoots, but has less insecticidal activity than the strain B. thuringiensis B-5689, which exhibits high insecticidal activity and colonizes mainly plant roots (25.37 ± 3.82 CFU × 105/g), reduced colonization of potato crops by the Colorado potato beetle, and also increased the yield of tubers in a 2-year experiment (2020–2021). Under the influence of B. thuringiensis B-5351, a decrease in the number of larvae of early age was observed, in contrast to B. thuringiensis B-5689, which caused longer metamorphosis processes. Apparently, the effect of B. thuringiensis B-5351 was the death of insects. It is important that processing B. thuringiensis B-5351 contributed to a significant decrease in the number of late-instar larvae on crops treated with this strain, as well as the degree of defoliation of plants by the pest, which was not observed with the action of B. thuringiensis B-5689. On plots treated with B. thuringiensis B-5351, the yield of commercial tubers and the total yield increased. A method is proposed for studying the endophytic potential of strains in relation to the terrestrial part of plants to search for biocontrol agents as a basis for creating algorithms for constructing microbiomes in agrocenoses.

Effect of minor gallium addition on corrosion, passivity, and antibacterial behaviour of novel β-type Ti–Nb alloys

Metastable Ti–Nb alloys are promising bone-implant materials due to improved mechanical biofunctionality and biocompatibility. To overcome increasing bacterial infection risk, alloying with antibacterial elements is a promising strategy. This study investigates the effect of minor gallium (Ga) additions (4, 8 wt% Ga) to as-cast and solution-treated β-type Ti–45Nb-based alloy (96(Ti–45Nb)-4Ga, 92(Ti–45Nb)-8Ga (wt.%)) on corrosion and passive film properties, as well as cytocompatibility and antibacterial activity. The electrochemical properties were evaluated by potentiodynamic polarization, electrochemical impedance spectroscopy (EIS), and Mott-Schottky analyses in phosphate-buffered saline (PBS). X-ray photoelectron spectroscopy (XPS) was performed to analyze the chemical composition of passive films. Early adhesion and viability of macrophages and Staphylococcus aureus were assessed by nucleocounting and colony-forming unit counting, respectively. The results showed that high corrosion resistance and passive film properties of Ti–45Nb are retained and even slightly improved with Ga. EIS results revealed that Ga addition improves the passive film resistance. XPS measurements of 92(Ti–45Nb)-8Ga show that the passive film contains Ti-, Nb- and Ga-based oxides, implying the formation of mixed (Ti–Nb-Ga) oxides. In addition, marginal Ga ion release rate was detected under free corrosion conditions. Therefore, it can be assumed that Ga species may contribute to passive film formation on Ga-containing alloys. The 92(Ti–45Nb)-8Ga elicited an antibacterial effect against S. aureus compared to cp-Ti at 4 h. Moreover, Ga-containing alloys showed good cytocompatibility with THP-1 macrophages at 24 h. In conclusion, it was demonstrated that Ga additions to Ti–45Nb are beneficial to corrosion resistance and showed promising initial host and bacterial interactions.

Next-generation Sequencing Results Require Higher Inoculum for Cutibacterium acnes Detection Than Conventional Anaerobic Culture.

Cutibacterium acnes has been described as the most common causative microorganism in prosthetic shoulder infections. Conventional anaerobic culture or molecular-based technologies are usually used for this purpose, but little to no concordance between these methodologies (k = 0.333 or less) has been observed. (1) Is the minimum C. acnes load for detection higher for next-generation sequencing (NGS) than for anaerobic conventional culture? (2) What duration of incubation is necessary for anaerobic culture to detect all C. acnes loads? Five C. acnes strains were tested for this study: Four strains were causing infection and were isolated from surgical samples. Meanwhile, the other was a reference strain commonly used as a positive and quality control in microbiology and bioinformatics. To create inoculums with varying degrees of bacterial load, we began with a standard bacterial suspension at 1.5 x 10 8 colony-forming units (CFU)/mL and created six more diluted suspensions (from 1.5 x 10 6 CFU/mL to 1.5 x 10 1 CFU/mL). Briefly, to do so, we transferred 200 µL from the tube with the highest inoculum (for example, 1.5 x 10 6 CFU/mL) to the following dilution tube (1.5 x 10 5 CFU/mL; 1800 µL of diluent + 200 µL of 1.5 x 10 6 CFU/mL). We serially continued the transfers to create all diluted suspensions. Six tubes were prepared per strain. Thirty bacterial suspensions were tested per assay. Then, 100 µL of each diluted suspension was inoculated into brain heart infusion agar with horse blood and taurocholate agar plates. Two plates were used per bacterial suspension in each assay. All plates were incubated at 37°C in an anaerobic chamber and assessed for growth after 3 days of incubation and daily thereafter until positive or Day 14. The remaining volume of each bacterial suspension was sent for NGS analysis to identify bacterial DNA copies. We performed the experimental assays in duplicate. We calculated mean DNA copies and CFUs for each strain, bacterial load, and incubation timepoint assessed. We reported detection by NGS and culture as a qualitative variable based on the identification or absence of DNA copies and CFUs, respectively. In this way, we identified the minimum bacterial load detected by NGS and culture, regardless of incubation time. We performed a qualitative comparison of detection rates between methodologies. Simultaneously, we tracked C. acnes growth on agar plates and determined the minimum incubation time in days required for CFU detection in all strains and loads examined in this study. Growth detection and bacterial CFU counting were performed by three laboratory personnel, with a high intraobserver and interobserver agreement (κ > 0.80). A two-tailed p value below 0.05 was considered statistically significant. Conventional cultures can detect C. acnes at a load of 1.5 x 10 1 CFU/mL, whereas NGS can detect bacteria when the concentration was higher, at 1.5 x 10 2 CFU/mL. This is represented by a lower positive detection proportion (73% [22 of 30]) for NGS than for cultures (100% [30 of 30]); p = 0.004). By 7 days, anaerobic cultures were able to detect all C. acnes loads, even at the lowest concentrations. When NGS is negative and culture is positive for C. acnes , there is likely a low bacterial load. Holding cultures beyond 7 days is likely unnecessary. This is important for treating physicians to decide whether low bacterial loads necessitate aggressive antibiotic treatment or whether they are more likely contaminants. Cultures that are positive beyond 7 days likely represent contamination or bacterial loads even below the dilution used in this study. Physicians may benefit from studies designed to clarify the clinical importance of the low bacteria loads used in this study at which both methodologies' detection differed. Moreover, researchers might explore whether even lower C. acnes loads have a role in true periprosthetic joint infection.

Polyethersulfone (PES) Filters Improve the Recovery of Legionella spp. and Enhance Selectivity against Interfering Microorganisms in Water Samples.

In the analysis of water samples, the type of filtration membrane material can influence the recovery of Legionella species, although this issue has been poorly investigated. Filtration membranes (0.45 µm) from different materials and manufacturers (numbered as 1, 2, 3, 4, and 5) were compared: mixed cellulose esters (MCEs), nitrocellulose (NC), and polyethersulfone (PES). After membrane filtration of samples, filters were placed directly onto GVPC agar and incubated at 36 ± 2 °C. The highest mean counts of colony-forming units and colony sizes for Legionella pneumophila and Legionella anisa were obtained with PES filters (p < 0.001). All membranes placed on GVPC agar totally inhibited Escherichia coli and Enterococcus faecalis ATCC 19443 and ATCC 29212, whereas only the PES filter from manufacturer 3 (3-PES) totally inhibited Pseudomonas aeruginosa. PES membrane performance also differed according to the manufacturer, with 3-PES providing the best productivity and selectivity. In real water samples, 3-PES also produced a higher Legionella recovery and better inhibition of interfering microorganisms. These results support the use of PES membranes in methods where the filter is placed directly on the culture media and not only in procedures where membrane filtration is followed by a washing step (according to ISO 11731:2017).

Assessing rhizosphere Trichoderma asperellum strains for root colonizing and antagonistic competencies against Fusarium wilt through molecular and biochemical responses in castor

The study was aimed at identifying the effective strain(s) of Trichoderma asperellum to colonize castor roots, promote growth and limit pathogen infection. Two castor genotypes; DCS-107 and 48-1 varying for stress tolerance to Fusarium wilt were treated with four Trichoderma asperellum strains; 7316, N13, TV5 and TV2B16 to analyze root colonizing and antagonistic competencies. A significant decline in the colony-forming unit (CFU) counts was observed in Trichoderma-treated bulk and rhizosphere soil compared to controls indicating a high rhizosphere competence of the Trichoderma asperellum strains; 7316 followed by N13. These strains also showed high mycoparasitic propensity against Fusarium oxysporum through dual culture-confrontation and in-planta assays. Bio-priming with Trichoderma strains enhanced plant growth in the tolerant genotype, DCS-107 compared to the susceptible genotype, 48-1 suggesting differential responses of the host genotypes. An increase in shoot length by 46.2, 28.9 and 21.7% was observed upon treatment of DCS-107 genotype with the Trichoderma strains, 7316, N13 and TV5 respectively. Further, a quantitative polymerase chain reactions (qPCRs) of 13 stress and defence response genes revealed gene transcript reprogramming, causing a transient repression of the plant immune responses to enable root colonization. Further, biochemical analysis revealed higher antioxidant enzyme activity of catalase, superoxide dismutase and glutathione peroxidase and total phenol content in co-stressed conditions compared to the controls suggesting that Trichoderma confers protection against oxidative stress damage. Trichoderma strains, 7316, N13 and TV5, either individually or in combination could be utilized as potential biocontrol agents to enhance growth and confer resistance to wilt pathogen, Fusarium oxysporum ricini.

Estimation of antimicrobial effect of non-thermal microwave plasma jet in model experiment

Relevance. The plasma treatment of ready-to-eat foods the potential use to increase a storage time and a food safety. On the first step seems important to evaluate the bactericidal effect of a non-thermal (non-equilibrium) plasma on a natural association bacteria isolated from the food.Methods. The non-thermal plasma source based on an argon plasma jet in a microwave (streamer) discharge at atmospheric pressure was used. Lactobacillus culture isolated from a walnut in the natural association and the vegetative form was treated. Sowing was carried on Petri dishes with Endoʼs agar. The exposure time is 60 s, 180 s and 360 s. The distance from the plasma jet source’s nozzle to the Endoʼs agar surface was 30 mm. The diameter of the growth inhibition zone of Lactobacillus on the dense nutrient medium was determined.Results. The round transparent Endo’s agar area — the zone of growth inactivation of colony-forming units in bacterial lawn of Lactobacillus after exposure to argon plasma jet was registered. After counting colony-forming units, a decrease in the number of Lactobacillus colonies grown after a 360-second exposure was found. At a 60-second exposure, no significant decrease in the number of grown colony-forming units was observed. The bactericidal effect is noticeable after a 180-second exposure to the plasma jet. With an increase in the exposure time to 360 s, the sterilization area on the surface of the agar in the Petri dish increased. The maximum diameter of the Lactobacillus growth inactivation zone did not exceed the diameter of the nozzle of the plasma jet source (36 mm). Under the influence of plasma, the color of the Endo agar changed in the zone of direct exposure to the plasma jet.

Viability of selected microorganisms in parenteral preparations for novel systemic anti-cancer therapy.

Risk factors for aseptic preparation of parenteral medicines encompass the growth-promoting nature of the preparation. Although many aqueous parenteral preparations do not have growth-promoting properties, inadvertently introduced microorganisms may remain viable. Knowledge about the viability of microorganisms in parenteral preparations can add useful information for assigning shelf life to preparations used to treat cancer patients. The aim of the study was to assess the viability of four different facultative pathogenic microorganisms in 20 ready-to-administer parenteral preparations aseptically prepared in hospital pharmacies. Samples of 20 different biologics and small molecules for systemic anti-cancer therapy were inoculated either with different bacteria (i.e., Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium) or with Candida albicans suspension. The resulting test concentrations were 104-105 microorganisms per mL. Aliquots of inoculated test solutions were transferred in duplicate to tryptic soy agar plates at the time points 0, 4, 24, 48, 144 h. The plates were incubated for 24 h (bacterial strains) and 72 h (C. albicans) at 37 °C and colony forming units (CFUs) were counted. In most test solutions, especially in monoclonal antibody solutions, increased CFU counts of P. aeruginosa and unchanged or increased CFU counts of E. faecium and S. aureus were registered. Pronounced nutritive properties of monoclonal antibodies and filgrastim were not registered. Azacitidine, pixantrone and vinflunine containing test solutions revealed species-specific bacteriostatic and even bactericidal activity. All test solutions, except nivolumab and pixantrone containing solutions, showed constant or increasing CFU counts of C. albicans after incubation. Viability of the selected pathogenic microorganisms was retained in most of the tested biological and small molecule preparations used to treat cancer patients. Therefore, in pharmacy departments strict aseptic conditions should be regarded and the lack of antimicrobial activity should be considered when assigning shelf life to RTA parenteral preparations.

Exploring the relationship between the reduction of floor microbial burden and the impact on healthcare-associated infections

Background: Healthcare floors are a vehicle and/or source for potential pathogens that cause healthcare associated infections, and hospital floors are often heavily contaminated with pathogens such as Clostridioides difficile and methicillin-resistant Staphylococcus aureus. However, definitive research linking reductions in floor burden to reductions in HAIs has not yet been established. We sought to evaluate emerging technology for continuous disinfection and its potential impact on HAIs. This study was designed to explore the potential relationship between the reduction of microbial burden of floors and healthcare associated infections. Methods: A prospective study was conducted in a 22-bed medical-surgical intensive care unit in a 180-bed suburban hospital near New Orleans, Louisiana, from November 2021 to June 2022. Using sterile, premoistened sponges, samples were collected from the floors of 10 areas throughout the unit including 2 nurses’ stations, the physician charting area, and 7 patient rooms. The advanced photocatalytic oxidation (aPCO) equipment was then installed in the HVAC ductwork throughout the ICU and activated. Environmental surface sampling of the same floor surfaces was then repeated every 4 weeks for the first 5 months of the study. HAIs were also tracked throughout the entire study period. The facility’s normal cleaning floor protocols using a neutralizing floor cleaner were unchanged and followed during the study. Changes in surface burden were calculated using a repeated-methods ANOVA with post hoc analyses as appropriate. Rates of healthcare associated infections were compared using χ2 analyses. Results: Overall, there was a 99.6% statistically significant decrease in floor environmental surface burden from the baseline to the final postactivation test (Fig. 1). The average colony forming unit count (CFU) decreased from 318,850 CFU per 100 cm2 to just 2,988 CFU per 100 cm2. The unit also saw a statistically significant decrease in publicly reported healthcare associated infections (HO-MRSA, CLABSI, HO-CDI) during the study period compared to the same period a year prior and in the 6 months immediately prior to the beginning of the study (Fig. 2). Conclusions: Advanced photocatalytic oxidation technology resulted in a reduction of microbial burden on the floors of a high-traffic intensive care unit. Statistically significant decreases in healthcare-associated infections was also seen. This study highlights a novel aPCO technology and its efficacy at reducing microbial burden and healthcare-associated infections despite no change in practice.Disclosures: None

Semi-automatic model to colony forming units counting

&lt;span lang="EN-US"&gt;Colony forming units counting is a conventional process carry out in bacteriological laboratories, and it is used to follow the behavior of bacteria in different conditions. Currently exist different systems, automatic or semi-automatic, to counting colony forming units exits, but, in general, many laboratories continue using manual counting, which consumes considerable time and effort from researchers and laboratory employees. This paper presents a mathematical model carry out to segment the colony forming units and, in this way, counting them from a digital image of the sample. The method uses the color space information of some points in the image and shows good behavior for images with many or few colony forming units in the sample, according to manual counting. The results show efficiencies close to 98% with MacConkey agar.&lt;/span&gt;

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.

A novel rodent model of chronic spinal implant-associated infection

BACKGROUND CONTEXTBacterial infection of spinal instrumentation is a significant challenge in spinal fusion surgery. Although the intraoperative local application of powdered vancomycin is common practice for mitigating infection, the antimicrobial effects of this route of administration are short-lived. Therefore, novel antibiotic-loaded bone grafts as well as a reliable animal model to permit the testing of such therapies are needed to improve the efficacy of infection reduction practices in spinal fusion surgery. PURPOSEThis study aims to establish a clinically relevant rat model of spinal implant-associated infection to permit the evaluation of antimicrobial bone graft materials used in spinal fusion. STUDY DESIGNRodent study of chronic spinal implant-associated infection. METHODSInstrumentation anchored in and spanning the vertebral bodies of L4 and L5 was inoculated with bioluminescent methicillin-resistant Staphylococcus aureus bacteria (MRSA). Infection was monitored using an in vivo imaging system (IVIS) for 8 weeks. Spines were harvested and evaluated histologically, and colony-forming units (CFUs) were quantified in harvested implants and spinal tissue. RESULTSPostsurgical analysis of bacterial infection in vivo demonstrated stratification between MRSA and phosphate-buffered saline (PBS) control groups during the first 4 weeks of the 8-week infection period, indicating the successful establishment of acute infection. Over the 8-week chronic infection period, groups inoculated with 1 × 105 MRSA CFU and 1 × 106 MRSA CFU demonstrated significantly higher bioluminescence than groups inoculated with PBS control (p = 0.009 and p = 0.041 respectively). Histological examination at 8 weeks postimplantation revealed the presence of abscesses localized to implant placement in all MRSA inoculation groups, with the most pervasive abscess formation in samples inoculated with 1 × 105 MRSA CFU and 1 × 106 MRSA CFU. Quantification of CFU plated from harvested spinal tissue at 8 weeks post-implantation revealed the 1 × 105 MRSA CFU inoculation group as the only group with a significantly greater average CFU count compared to PBS control (p = 0.017). Further, CFU quantification from harvested spinal tissue was greater than CFU quantification from harvested implants across all inoculation groups. CONCLUSIONOur model demonstrated that the inoculation dosage of 1 × 105 MRSA CFU exhibited the most robust chronic infection within instrumented vertebral bodies. This dosage had the greatest difference in bioluminescence signal from control (p < 0.01), the lowest mortality (0% compared to 50% for samples inoculated with 1 × 106 MRSA CFU), and a significantly higher amount of CFUs from harvested spine samples than CFUs from control harvested spine samples. Further, histological analysis confirmed the reliability of this novel rodent model of implanted-associated infection to establish infection and biofilm formation of MRSA for all inoculation groups. CLINICAL SIGNIFICANCEThis model is intended to simulate the infection of instrumentation used in spinal fusion surgeries concerning implant locality and material. This model may evaluate potential antimicrobial and osteogenic biomaterials and investigate the relationship between implant-associated infection and failed fusion.

Efficacy of adjunct photodynamic therapy on Candida growth and oral health quality of life in denture stomatitis patients with type 2 diabetes mellitus wearing implant-retained overdentures: A randomized clinical study

AimThe aim of the present clinical trial was to study the efficacy of combined miconazole and PDT in the improvement of quality of life and levels of Candida species in chronic hyperglycemic patients with denture stomatitis (DS). MethodsOne hundred patients were randomly divided into five groups; 20 each in the miconazole, PDT, miconazole+ PDT, CHX and distilled water groups. Methylene blue mediated irradiation was conducted using 600 nm diode laser with power, energy density and radiance as 100 mW, 3527 mW/cm2 and 9 J, respectively. Patients were advised to apply 2.5 ml of 2% topical miconazole four times a day. The existence of Candida spp. was detected by means of microbiological culture technique. Candida colony counts from the palates and dentures surfaces, quantified as colony forming unit (CFU)/mL were evaluated at baseline, end of 14 days, 28 days and 60 days. Oral health related quality of life was assessed with the help of a questionnaire. ResultsThe quality of life showed significant improvement in the group where combination treatment was executed. The overall CFU/mL values were greater in the dentures in comparison to those from the palates of the patients of all the five groups. During all time periods of the study, the CFU/mL values obtained from combination treatment group showed significant differences. Candida albicans was the most predominant yeast. ConclusionThis study showed the effectiveness of methylene blue- PDT in combination with miconazole in improving oral health related quality of life and significantly reducing Candida CFU counts to resolve palatal inflammation in diabetic individuals with implant-supported complete dentures.

Enhancing antimicrobial photodynamic therapy with phenothiazinium dyes and sodium dodecyl sulfate against Candida albicans at various growth stages

BackgroundThe eradication of C. albicans is difficult due to the organization of the yeast in biofilms. Photodynamic therapy (PDT) has been proposed as an alternative to antifungals. Phenothiazinium dyes, e.g. methylene blue (MB), have been proposed as photosensitizing agents (PS), and their association with sodium dodecyl sulfate (SDS) has recently been shown to improve the effectiveness of PDT in planktonic culture. In this sense, the objective of this work was to evaluate the effect of PDT with phenothiazinium dyes associated to SDS in biofilms at the different stages of growth. MethodsExperiments were carried out to evaluate the effects of PDT on biofilm formation and on established biofilms of C. albicans ATCC 10231. Samples were exposed to PS 50 mg/L (MB, Azure A – AA, Azure B - AB and dimethyl methylene blue - DMMB) dissolved in water or 0.25% SDS, for 5 min in the dark. After irradiation at 660 nm, 37.3mW/cm2 for 27 min, 60.4J/cm2 colony forming units count assay (CFU/mL) was performed. One or two irradiations were applied. Statistical methods were used to assess effectiveness. ResultsPSs showed low toxicity in the dark. An application of PDT irradiation was not able to reduce the CFU/mL both in mature biofilms (24h) and in biofilms in the dispersion phase (48h), only in the adherence phase did PDT prevent the formation of biofilms. With two successive applications of PDT irradiation in the dispersion phase, PDT with MB, AA, and DMMB completely inactivated C. albicans. The similar was not observed with mature biofilms. ConclusionsDifferent stages of biofilm growth respond differently to PDT, with the greatest inhibitory effect found in the adhesion stage. Mature and dispersed biofilms are less susceptible to PDT. The use of two successive applications of PDT with PSs associated with SDS may be a useful approach to inactivate C. albicans biofilms.

In vitro and in vivo evaluation of diethyldithiocarbamate with copper ions and its liposomal formulation for the treatment of Staphylococcus aureus and Staphylococcus epidermidis biofilms

Surgical site infections (SSIs) are mainly caused by Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) biofilms. Biofilms are aggregates of bacteria embedded in a self-produced matrix that offers protection against antibiotics and promotes the spread of antibiotic-resistance in bacteria. Consequently, antibiotic treatment frequently fails, resulting in the need for alternative therapies. The present study describes the in vitro efficacy of the Cu(DDC)2 complex (2:1 M ratio of diethyldithiocarbamate (DDC−) and Cu2+) with additional Cu2+ against S. aureus and S. epidermidis biofilms in models mimicking SSIs and in vitro antibacterial activity of a liposomal Cu(DDC)2 + Cu2+ formulation. The in vitro activity on S. aureus and S. epidermidis biofilms grown on two hernia mesh materials and in a wound model was determined by colony forming unit (CFU) counting. Cu2+-liposomes and Cu(DDC)2-liposomes were prepared, and their antibacterial activity was assessed in vitro using the alamarBlue assay and CFU counting and in vivo using a Galleria mellonella infection model. The combination of 35 μM DDC− and 128 μM Cu2+ inhibited S. aureus and S. epidermidis biofilms on meshes and in a wound infection model. Cu(DDC)2-liposomes + free Cu2+ displayed similar antibiofilm activity to free Cu(DDC)2 + Cu2+, and significantly increased the survival of S. epidermidis-infected larvae. Whilst Cu(DDC)2 + Cu2+ showed substantial antibiofilm activity in vitro against clinically relevant biofilms, its application in mammalian in vivo models is limited by solubility. The liposomal Cu(DDC)2 + Cu2+ formulation showed antibiofilm activity in vitro and antibacterial activity and low toxicity in G. mellonella, making it a suitable water-soluble formulation for future application on infected wounds in animal trials.

Enhanced in vitro antibacterial effect against Enterococcus faecalis by using both low-dose cetylpyridinium chloride and silver ions

BackgroundEnterococcus faecalis (E. faecalis) is frequently isolated from root canals with failed root canal treatments. Due to the strong ability of E. faecalis to resist many often-used antimicrobials, coping with E. faecalis infections remains a challenge. The aim of this study was to investigate the synergistic antibacterial effect of low-dose cetylpyridinium chloride (CPC) and silver ions (Ag+) against E. faecalis in vitro.MethodsThe minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and the fractional inhibitory concentration index (FICI) were used to confirm the existence of the synergic antibacterial activity between low-dose CPC and Ag+. Colony-forming unit (CFU) counting, time-killing curve and dynamic growth curve were used to evaluate the antimicrobial effects of CPC and Ag+ combinations against planktonic E. faecalis. Four weeks biofilms were treated with drug-contained gels to determine the antimicrobial effect on biofilm-resident E.faecalis, and the integrity of E.faecalis and its biofilms were observed by FE-SEM. CCK-8 assays was used to test the cytotoxicity of CPC and Ag+ combinations on MC3T3-E1 cells.ResultsThe results confirmed the synergistic antibacterial effect of low-dose CPC and Ag+ against both planktonic and 4-week biofilm E. faecalis. After the addition of CPC, the sensitivity of both planktonic and biofilm-resident E. faecalis to Ag+ improved, and the combination showed good biocompatibility on MC3T3-E1 cells.ConclusionsLow-dose CPC enhanced the antibacterial ability of Ag+ against both planktonic and biofilm E.faecalis with good biocompatibility. It may be developed into a novel and potent antibacterial agent against E.faecalis, with low toxicity for root canal disinfection or other related medical applications.

Ozonated Water Promotes in vitro Decontamination of Dental Implants Surface

ABSTRACT Although studies have shown high survival rates of dental implants, complications may occur mainly due to peri-implant infections. These highlight the importance of establishing protocols for implant decontamination using adjuvant therapies, such as ozone. This study aimed to evaluate the potential for in vitro decontamination of ozonated water on implants surface. Sixteen titanium implants were used, distributed into the groups (n = 4): group E – sterile implants; group C – contaminated implants; group O40 – contaminated implants treated with ozonated water 40 mg/L; and group O60 – contaminated implants treated with ozonated water 60 mg/L. The implants of groups C, O40, and O60 were contaminated in vitro with biofilm. Groups O40 and O60 were decontaminated with ozonated water for 1 minute. Microbiologic analysis was performed by counting colony-forming units (CFUs). Experimental group comparisons were done using a one-way ANOVA test with Tukey’s posttest; a comparison between groups O40 and O60 was done using an independent t-test (p < 0.05). There was a difference among all experimental groups (p = 0.000), with a more significant reduction in CFUs in group O60 (0.83 × 107 ± 0.06) compared to group C (3.41 × 107 ± 0.41). It was concluded that ozonated water 60 mg/L has shown good potential for in vitro decontamination of implants surface.

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.