Microbial Bioburden Research Articles

Raman spectroscopy as an alternative rapid microbial bioburden test method for continuous, automated detection of contamination in biopharmaceutical drug substance manufacturing.

To investigate an in-line Raman method capable of detecting accidental microbial contamination in pharmaceutical vessels, such as bioreactors producing monoclonal antibodies via cell culture. The Raman method consists of a multivariate model built from Raman spectra collected in-line during reduced-scale bioreactor batches producing a monoclonal antibody, as well as a reduced-scale process with intentional spiking of representative compendial method microorganisms (n=4). The orthogonal partial least squares regression discriminant analysis model (OPLS-DA) area under the curve (AUC), specificity and sensitivity were 0.96, 0.99, and 0.95, respectively. Furthermore, the model successfully detected contamination in an accidentally contaminated manufacturing-scale batch. In all cases, the time to detection (TTD) for Raman was superior compared to offline, traditional microbiological culturing. The Raman OPLS-DA method met acceptance criteria for equivalent decision making to be considered a viable alternative to the compendial method for in-process bioburden testing. The in-line method is automated, non-destructive, and provides a continuous assessment of bioburden compared to an offline compendial method, which is manual, results in loss of product, and in practice is only collected once daily and requires 3-5 days for enumeration.

Trends in viable microbial bioburden on surfaces within a paediatric bone marrow transplant unit

Despite their role being historically overlooked, environmental surfaces have been shown to play a key role in the transmission of pathogens causative of healthcare-associated infection. To guide infection prevention and control (IPC) interventions and inform clinical risk assessments, more needs to be known about microbial surface bioburdens. To identify the trends in culturable bacterial contamination across communal touch sites over time in a hospital setting. Swab samples were collected over nine weeks from 22 communal touch sites in a paediatric bone marrow transplant unit. Samples were cultured on Columbia blood agar and aerobic colony counts (ACC) per 100cm2 were established for each site. Individual colony morphologies were grouped and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or 16s rDNA sequencing. Highest mean counts were observed for sites associated with ward management activity and computer devices (3.29 and 2.97 ACC/100cm2 respectively). A nurses' station keyboard had high mean ACC/100cm2 counts (10.67) and diversity, while laundry controls had high mean ACC/100cm2 counts (4.70) and low diversity. Micrococcus luteus was identified in all sampling groups. Clinical staff usage sites were contaminated with similar proportions of skin and environmental flora (52.19-46.59% respectively), but sites associated with parental activities were predominantly contaminated by environmental microflora (86.53%). The trends observed suggest patterns in microbial loading based on site activities, surface types and user groups. Improved understanding of environmental surface contamination could help support results interpretation and IPC interventions, improving patient safety.

A Diagnostic-Driven Prospective Clinical Study Evaluating the Combination of an Antibiofilm Agent and Negative Pressure Wound Therapy.

Each year, millions of Americans develop truncal pressure ulcers (PUs) which can persist for months, years, or until the end of life. Despite the negative impact on quality of life and escalating costs associated with PUs, there is sparse evidence supporting validated and efficacious treatment options. As a result, treatment is based on opinion and extrapolation from other wound etiologies. The ideal reconstructive plan maximizes the patient's nutritional status, incorporates the basic tenets of wound bed preparation (debridement, offloading, proper moisture balance, reduction of bacterial burden), and employs diagnostics to guide therapeutic intervention. The use of combination therapies can potentially overcome several of the barriers to wound healing. Negative pressure wound therapy (NPWT), a commonly used modality in the management of PUs, facilitates healing by stimulating the formation of granulation tissue and promoting wound contraction; however, NPWT alone is not always effective. Clinical studies examining microbial bioburden in PUs determined that most ulcers contain bacteria at levels that impede wound healing (>104 CFU/g). Thus, we hypothesized that adding an anti-microbial agent to decrease both planktonic and biofilm bacteria in the wound would increase the efficacy of NPWT. In this prospective study, twenty patients with recalcitrant PUs that previously failed NPWT were treated with a biofilm-disrupting agent (Blast-X, Next Science, Jacksonville, FL, USA) in combination with NPWT. Fluorescence imaging was used to follow bacterial burden and guide therapy. In total, 45% of the PUs reduced in size over the course of the four-week study, with a resolution of bacterial fluorescence in the NPWT dressing and wound bed seen in an average of three weeks. The combination of an antibiofilm agent and NPWT reduced bacterial levels and improved wound healing in recalcitrant PUs.

Real-World Evidence of the Impact of a Novel Surgical Irrigant on Surgical Site Infections in Primary Total Knee Arthroplasty Performed at an Ambulatory Surgery Center.

Background: Total knee arthroplasty (TKA) is one of the most common inpatient and outpatient surgical procedures performed in the United States and is predicted to increase 401% by 2040. Surgical site infections (SSIs) at an incidence rate of approximately 2% are costly post-operative complications in TKA. Intra-operative surgical irrigants are used to decrease contaminating microbial bioburden within the surgical site to prevent SSI. The primary objective of this retrospective study was to evaluate the impact of a novel surgical irrigant called XPERIENCE® Advanced Surgical Irrigation (XP; Next Science, Jacksonville, FL) on SSI incidence in primary TKA performed at an ambulatory surgery center (ASC). Patients and Methods: Primary TKAs were performed at a free-standing ASC. The novel surgical irrigant was used intra-operatively to rinse away debris and micro-organisms from the surgical site. Retrospective data collation included SSI rates, complication rates, and re-admissions due to SSI within 90 days of index surgery. Results: Among the 524 primary TKA surgeries, one peri-prosthetic joint infection (PJI) was diagnosed within 90 days of index surgery and one superficial incisional SSI was diagnosed within 30 days of index surgery. The PJI was attributed to an exogenously acquired upper respiratory tract infection rather than due to the failure of intra-operative regimes. The 0.19% PJI incidence rate indicated significant efficacy of XP in decreasing PJI. An overall complication rate of 7.82% was noted with none of the complications associated with usage of the novel surgical irrigant. Conclusions: XPERIENCE is a promising intra-operative antimicrobial irrigant that can be easily incorporated into a broader infection prevention strategy.

Anti-bacterial and anti-inflammatory properties of Vernonia arborea accelerate the healing of infected wounds in adult Zebrafish.

Management of wounds and healing under impaired conditions are the major challenges faced globally by healthcare workers. Phytocompounds which are anti-microbial and capable of modulating inflammation contribute to overall wound healing and regain of the lost structure and function especially in wounds impaired with polymicrobial infection. An acute cutaneous impaired wound model using adult zebrafish was validated to simulate mammalian wound pathophysiology. This model was used to evaluate phytofractions of Vernonia arborea in the present study, for reduction of infection; myeloperoxidase (MPO) as a marker of infection; neutrophil infiltration and resolution; kinetics of inflammatory cytokines; and wound repair kinetics (viz., nitrite levels and iNoS expression; reepithelisation). Four fractions which were active in-vitro against five selected wound microbes were shown to reduce ex-vivo microbial bioburden upto 96% in the infected wound tissue. The reduction in CFU correlated with the neutrophil kinetics and MPO enzyme levels in the treated, wound infected zebrafish. Expression of pro-inflammatory cytokines (IL-6 and TNF-α) was downregulated while upregulating anti-inflammatory cytokine (IL-10), and nitric oxide signalling with fourfold increase in iNOS expression. The adult zebrafish wound model could well serve as a standard tool for assessing phytoextracts such as V. arborea for wound healing with anti-microbial properties.

Risk-based approach to setting sterile filtration microbial bioburden limits – Focus on biotech-derived products

Holistic concepts should be applied that reduce risks prior to final bioburden testing and sterile filtration, based on enhanced process and product attribute understanding, which could be key to successful bioburden risk management.Key findings of this paper include:oBioburden control and monitoring prior to filter sterilization is necessary for any thermolabile parenteral drug product that cannot be terminally sterilized in the final container. The bioburden limit applied prior to filter sterilization, as recommended in the 2019 EMA guideline “The sterilisation of the medicinal product, active substance, excipient and primary container”, can be interpreted as a specification (i.e., pass/fail criterion) regardless of the sterilizing filter capacity and risk mitigation measures applied during manufacture. It is proposed to interpret the guidance given by the EMA as an action limit of ≤10 CFU/100 mL.oPre-filtration and post-filtration risks and control measures are inter-correlated and should be addressed via an integrated risk assessment. This includes selection of sample test volume, acceptable bioburden levels and sterilizing filter area.oA holistic approach to bioburden risk is recommended, using a two-tier system for low bioburden monitoring prior to sterilization. A low alert level (product/facility history) and a higher action level, at or below the EMA criterion would be appropriate. It is proposed that any excursion warrants a thorough investigation and a case-by-case assessment for product quality impact rather than default rejection.oThe paper describes how detectable bioburden may be mitigated through subsequent effective sterilizing filtration, if the potential for bacterial by-products and their impact on safety and protein stability is assessed.oAs the manufacturing process is highly controlled to limit the potential for bioburden growth prior to filter sterilization, sample volumes less than 100 mL and alternative acceptance levels to ≤10 CFU/100 mL can be justified on the basis of the actual sample subject to testing. This is particularly relevant for small volume product batches.oTesting of bioburden prior to sterile filtration is not the only measure to control the microbial status and to generate sterile product but is a monitoring tool to verify efficacy of the control measures.

Immersion of debrided diabetic foot ulcer tissue in electrochemically generated pH neutral hypochlorous acid significantly reduces the microbial bioburden: whole-genome sequencing of Staphylococcus aureus, the most prevalent species recovered

Diabetic foot ulcer infections (DFUIs) are the leading cause of lower-limb amputations, mediated predominantly by Staphylococcus aureus. pH-neutral electrochemically generated hypochlorous acid (anolyte) is a non-toxic, microbiocidal agent with significant potential for wound disinfection. To investigate both the effectiveness of anolyte for microbial bioburden reduction in debrided ulcer tissues and the population of resident S.aureus. Fifty-one debrided tissues from 30 people with type II diabetes were aliquoted by wet weight and immersed in 1- or 10-mL volumes of anolyte (200parts per million) or saline for 3min. Microbial loads recovered were determined in colony forming units/g (cfu/g) of tissue following aerobic, anaerobic and staphylococcal-selective culture. Bacterial species were identified and 50S.aureus isolates from 30 tissues underwent whole-genome sequencing (WGS). The ulcers were predominantly superficial, lacking signs of infection (39/51, 76.5%). Of the 42/51 saline-treated tissues yielding ≥105cfu/g, a microbial threshold reported to impede wound-healing, only 4/42 (9.5%) were clinically diagnosed DFUIs. Microbial loads from anolyte-treated tissues were significantly lower than saline-treated tissues using 1mL (1065-fold, 2.0 log) and 10mL (8216-fold, 2.1 log) immersion volumes (P<0.0005). S.aureus was the predominant species recovered (44/51, 86.3%) and 50 isolates underwent WGS. All were meticillin susceptible and comprised 12 sequence types (STs), predominantly ST1, ST5 and ST15. Whole-genome multi-locus sequence typing identified three clusters of closely related isolates from 10 patients indicating inter-patient transmission. Short immersions of debrided ulcer tissue in anolyte significantly reduced microbial bioburden: a potential novel DFUI treatment.

Efficacy of a filtered far-UVC handheld disinfection device in reducing the microbial bioburden of hospital surfaces

The filtered far-UV-C (FFUV) handheld disinfection device is a small portable device that emits far UV-C at 222nm. The objective of this study was to evaluate the device's ability to kill microbial pathogens on hospital surfaces and compare it to manual disinfection using germicidal sodium hypochlorite wipes. A total of 344 observations (4 observations from 86 objects' surfaces) were sampled with 2 paired samples per surface: a pre- and a post-sodium hypochlorite and FFUV sample. The results were analyzed via a Bayesian multilevel negative binomial regression model. The estimated mean colony counts for the sodium hypochlorite control and treatment groups were 20.5 (95% uncertainty interval:11.7-36.0) and 0.1 (0.0-0.2) colony forming units (CFUs), respectively. The FFUV control and treatment groups had mean colony counts of 22.2 (12.5-40.1) and 4.1 (2.3-7.2) CFUs. The sodium hypochlorite group and the FFUV group had an estimated 99.4% (99.0%-99.7%) and 81.4% (76.2%-85.7%) reduction in colony counts, respectively. The FFUV handheld device effectively reduced the microbial bioburden on surfaces in the health care setting. The major benefit of FFUV is likely seen when manual disinfection is not possible or when supplementing cleaners or disinfectants with the low-level disinfection properties.

Metagenomic Methods for Addressing NASA's Planetary Protection Policy Requirements on Future Missions: A Workshop Report.

Molecular biology methods and technologies have advanced substantially over the past decade. These new molecular methods should be incorporated among the standard tools of planetary protection (PP) and could be validated for incorporation by 2026. To address the feasibility of applying modern molecular techniques to such an application, NASA conducted a technology workshop with private industry partners, academics, and government agency stakeholders, along with NASA staff and contractors. The technical discussions and presentations of the Multi-Mission Metagenomics Technology Development Workshop focused on modernizing and supplementing the current PP assays. The goals of the workshop were to assess the state of metagenomics and other advanced molecular techniques in the context of providing a validated framework to supplement the bacterial endospore-based NASA Standard Assay and to identify knowledge and technology gaps. In particular, workshop participants were tasked with discussing metagenomics as a stand-alone technology to provide rapid and comprehensive analysis of total nucleic acids and viable microorganisms on spacecraft surfaces, thereby allowing for the development of tailored and cost-effective microbial reduction plans for each hardware item on a spacecraft. Workshop participants recommended metagenomics approaches as the only data source that can adequately feed into quantitative microbial risk assessment models for evaluating the risk of forward (exploring extraterrestrial planet) and back (Earth harmful biological) contamination. Participants were unanimous that a metagenomics workflow, in tandem with rapid targeted quantitative (digital) PCR, represents a revolutionary advance over existing methods for the assessment of microbial bioburden on spacecraft surfaces. The workshop highlighted low biomass sampling, reagent contamination, and inconsistent bioinformatics data analysis as key areas for technology development. Finally, it was concluded that implementing metagenomics as an additional workflow for addressing concerns of NASA's robotic mission will represent a dramatic improvement in technology advancement for PP and will benefit future missions where mission success is affected by backward and forward contamination.

The relationship between microbial community vitality and ATP bioburden in bottom waters under fuel microcosms.

Adenylate energy charge (AEC) - computed from the ATP, ADP and AMP concentrations in a specimen - reflect the net physiological state of the microbial population in that specimen. Previous research has demonstrated that healthy microbial populations maintain AEC≥0.8. As populations are subjected to stresses, or - in closed systems - deplete the available nutrients, respond to the accumulation of toxic metabolites, or both, AEC decreases (often to <0.5). Aqueous-phase samples from a set of fuel-water microcosms were tested for cellular ATP (cATP) and AEC. This paper reports on the precision of the AEC test method and the relationship between cellular AEC and cATP bioburdens in the aqueous phase of fuel over aqueous-phase microcosms.

Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies

A well-validated and holistic program that incorporates robust gowning, cleaning, environmental monitoring, and personnel monitoring measures is critical for minimizing the microbial bioburden in cellular therapy manufacturing suites and the corresponding testing laboratories to ensure that the facilities are operating in a state of control. Ensuring product safety via quality control measures, such as sterility testing, is a regulatory requirement for both minimally manipulated (section 361) and more than minimally manipulated (section 351) human cells, tissues, and cellular and tissue-based products (HCT/Ps). In this video, we provide a stepwise guide for how to develop and incorporate the best aseptic practices for operating in a cleanroom environment, including gowning, cleaning, staging of materials, environmental monitoring, process monitoring, and product sterility testing using direct inoculation, provided by the United States Pharmacopeia (USP<71>) and the National Institutes of Health (NIH) Alternative Sterility Testing Method. This protocol is intended as a reference guide for establishments expected to meet current good tissue practices (cGTP) and current good manufacturing practices (cGMP).

Investigation of microorganisms in cannabis after heating in a commercial vaporizer.

There are concerns about microorganisms present on cannabis materials used in clinical settings by individuals whose health status is already compromised and are likely more susceptible to opportunistic infections from microbial populations present on the materials. Most concerning is administration by inhalation where cannabis plant material is heated in a vaporizer, aerosolized, and inhaled to receive the bioactive ingredients. Heating to high temperatures is known to kill microorganisms including bacteria and fungi; however, microbial death is dependent upon exposure time and temperature. It is unknown whether the heating of cannabis at temperatures and times designated by a commercial vaporizer utilized in clinical settings will significantly decrease the microbial loads in cannabis plant material. To assess this question, bulk cannabis plant material supplied by National Institute on Drug Abuse (NIDA) was used to assess the impact of heating by a commercial vaporizer. Initial method development studies using a cannabis placebo spiked with Escherichia coli were performed to optimize culture and recovery parameters. Subsequent studies were carried out using the cannabis placebo, low delta-9 tetrahydrocannabinol (THC) potency and high THC potency cannabis materials exposed to either no heat or heating for 30 or 70 seconds at 190°C. Phosphate-buffered saline was added to the samples and the samples agitated to suspend the microorganism. Microbial growth after no heat or heating was evaluated by plating on growth media and determining the total aerobic microbial counts and total yeast and mold counts. Overall, while there were trends of reductions in microbial counts with heating, these reductions were not statistically significant, indicating that heating using standard vaporization parameters of 70 seconds at 190°C may not eliminate the existing microbial bioburden, including any opportunistic pathogens. When cultured organisms were identified by DNA sequence analyses, several fungal and bacterial taxa were detected in the different products that have been associated with opportunistic infections or allergic reactions including Enterobacteriaceae, Staphylococcus, Pseudomonas, and Aspergillus.

Current research on fungi in chronic wounds.

The occurrence of chronic wounds is a major global health issue. These wounds are difficult to heal as a result of disordered healing mechanisms. The most common types of chronic wounds are diabetic ulcers, pressure ulcers, arterial/venous ulcers and nonhealing surgical wounds. Although bacteria are an important cause of chronic nonhealing wounds, fungi also play a substantial role in them. The fungal infection rate varies with different chronic wound types, but overall, the prevalence of fungi is extremely underestimated in the clinical treatment and management of chronic wounds. Wounds and ulcers can be colonized by host cutaneous, commensal or environmental fungi and evolve into local infections, causing fungemia as well as invasive fungal disease. Furthermore, the fungi involved in nonhealing wound-related infections help commensal bacteria resist antibiotics and the host immune response, forcing wounds to become reservoirs for multiresistant species, which are considered a potential key factor in the microbial bioburden of wounds and ulcers. Fungi can be recalcitrant to the healing process. Biofilm establishment is the predominant mechanism of fungal resistance or tolerance to antimicrobials in chronic nonhealing wounds. Candida albicans yeast and Trichophyton rubrum filamentous fungi are the main fungi involved in chronic wound infection. Fungal species diversity and drug resistance phenotypes in different chronic nonhealing wound types will be emphasized. In this review, we outline the latest research on fungi in chronic wounds and discuss challenges and future perspectives related to diagnosing and managing chronic wounds.

Assessment of microbial bioburden on portable medical equipment in a hospital setting.

Although routine disinfection of portable medical equipment is required in most hospitals, frontline staff may not be able to disinfect portable medical equipment at a rate that adequately maintains low bioburden on high-use equipment. This study quantified bioburden over an extended time period for two types of portable medical equipment, workstations on wheels and vitals machines, across three hospital wards. Bioburden was quantified via press plate samples taken from high touch surfaces on 10 workstations on wheels and 5 vitals machines on each of 3 medical surgical units. The samples were taken at three timepoints each day over a 4-week period, with random rotation of timepoints and portable medical equipment, such that frontline staff were not aware at which timepoint their portable medical equipment would be sampled. The mean bioburden from the different locations and portable medical equipment was estimated and compared with Bayesian multilevel negative binomial regression models. Model estimated mean colony counts (95% credible interval) were 14.4 (7.7-26.7) for vitals machines and 29.2 (16.1-51.1) for workstations on wheels. For the workstations on wheel, colony counts were lower on the mouse, 0.22 (0.16-0.29), tray, 0.29 (0.22, 0.38), and keyboard, 0.43 (0.32-0.55), when compared to the arm, as assessed by incident rate ratios. Although routine disinfection is required, bioburden is still present across portable medical equipment on a variety of surfaces. The difference in bioburden levels among surfaces likely reflects differences in touch patterns for the different portable medical equipment and surfaces on the portable medical equipment. Although the association of portable medical equipment bioburden to healthcare-associated infection transmission was not assessed, this study provides evidence for the potential of portable medical equipment as a vector for healthcare-associated infection transmission despite hospital disinfection requirements.

Performance equivalence and validation of a rapid microbiological method for detection and quantification of yeast and mold in an antacid oral suspension.

Alternative and rapid microbiological methods are effective and profitable technologies that can be safely use in the pharmaceutical industry. This report studies the efficacy of the Soleris® direct yeast and mold automated method for the quantitative detection of C. albicans and A. brasiliensis at three different microbial bioburden. Validation testing was carried out using an antacid oral suspension (aluminum hydroxide 4% + magnesium hydroxide 4% + simethicone 0.4%). Equivalence of data between detection time vs. colony-forming units was established for both, alternative and conventional methodologies. Using probability of detection, linear Poisson regression, Fisher's test, and multifactorial analysis of variance ANOVA, all results from the rapid method were prove to be in statistical agreement with the reference plating procedures. The limits of detection and quantification were statistically similar for both methods (Fisher's exact test, P >0.05), showing that the alternative method was not inferior in performance to the reference method. Essential validation parameters such as precision (standard deviation < 5, coefficient of variance < 35%), accuracy (>70%), linearity (R2 >0.9025), and ruggedness (ANOVA, P <0.05) were determined. It was proved that all test results obtained from the alternative method were in statistical agreement with the standard plate count method. Thus, this new technology was found to meet all the validation criteria needed to be considered as an alternative method for yeast and mold quantification in the antacid oral suspension tested.

Potential for Microbial Cross Contamination of Laundry from Public Washing Machines

Although clothes washing machines remove dirt, microorganisms are not reliably removed by modern cold-water machine-washing practices. Microbial bioburden on clothing originates from the wearer’s skin, the environment (indoor and outdoor), and the washing machine itself. While most clothing microbes are commensals, microbes causing odors and opportunistic pathogens may also be present. Understanding the extent of microbial transfer from washing machines to clothes may inform strategies for odor control and for mitigating the transmission of microbes through the laundering process. This study was designed to quantify and identify bacteria/fungi transferred from laundromat machines to sentinel cotton washcloths under standard cold-water conditions. Bacterial 16S rRNA and fungal ITS sequencing enabled identification of microorganisms in the washcloths following laundering. Total plate-based enumeration of viable microorganisms also was performed, using growth media appropriate for bacteria and fungi. Opportunistic human bacterial pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., were recovered. The fungal bioburden was ~two-fold lower than the bacterial bioburden. Most sequences recovered were assigned to non-pathogenic fungi, such as those from genera Malassezia and Ascomycota. These results suggest that public washing machines represent a source of non-pathogenic and pathogenic microbial contamination of laundered garments.

A Review of Immunomodulatory Reprogramming by Probiotics in Combating Chronic and Acute Diabetic Foot Ulcers (DFUs).

Diabetic foot ulcers (DFUs) are characterized by a lack of angiogenesis and distal limb diabetic neuropathy. This makes it possible for opportunistic pathogens to protect the biofilm-encased micro-communities, causing a delay in wound healing. The acute and chronic phases of DFU-associated infections are distinguished by the differential expression of innate proinflammatory cytokines and tumor necrosis factors (TNF-α and -β). Efforts are being made to reduce the microbial bioburden of wounds by using therapies such as debridement, hyperbaric oxygen therapy, shock wave therapy, and empirical antibiotic treatment. However, the constant evolution of pathogens limits the effectiveness of these therapies. In the wound-healing process, continuous homeostasis and remodeling processes by commensal microbes undoubtedly provide a protective barrier against diverse pathogens. Among commensal microbes, probiotics are beneficial microbes that should be administered orally or topically to regulate gut-skin interaction and to activate inflammation and proinflammatory cytokine production. The goal of this review is to bridge the gap between the role of probiotics in managing the innate immune response and the function of proinflammatory mediators in diabetic wound healing. We also highlight probiotic encapsulation or nanoformulations with prebiotics and extracellular vesicles (EVs) as innovative ways to tackle target DFUs.

Assessment of the Sterility of New Endodontic Files Received From the Manufacturer Using Microbial Culture and Scanning Electron Microscopic Analysis: An In Vitro Study.

BackgroundAs endodontic hand files are essential tools for root canal instrumentation and treatment, these instruments have been a constant source of debate regarding whether to reuse them or use them just once. The aim of this in vitro study is to evaluate the sterility of five brands’ new endodontic files received from manufacturers without sterilization before clinical use via microbial culture (bacteria and fungi) and bioburden using scanning electron microscopic (SEM) analysis.MethodologyThis study included 25 K-type hand files with equal numbers from five brand groups (n=5; groups 1 to 5), named Tia Dent, Prevalent, Mani, Thomas, and Dentsply, respectively. The new files were exposed to microbial culture prior to clinical use and SEM analyses. A Fisher’s exact test was performed to compare the differences in contamination among the five tested groups.ResultsIn the SEM, all the five brand groups showed one contaminated file out of five files (20%). Fisher's exact test findings revealed statistically significant variations between the five brand groups, as nine out of 25 (36%) files of the tested groups had positive contamination in the microbial culture experiment. Bacterial contamination was present in three (60%) of the five Tia Dent group files, four (80%) in the Prevalent group files, and two (40%) in the Mani group files. Those with fungal contamination were one (33%) in the Tia Dent group and one (25%) in the Prevalent group (P=0.027). The bacterial culture showed that 100% of the new K-files tested negative in both the Thomas and Dentsply groups.ConclusionsThis study concluded that there was a bioburden on new endodontic K-type hand files of different brand groups before sterilization or when immediately used.

An Engineered Nanocomposite Copper Coating with Enhanced Antibacterial Efficacy

AbstractContaminated surfaces are a major source of nosocomial infection. To reduce microbial bioburden and surface‐based transmission of infectious disease, the use of antibacterial and self‐sanitizing surfaces, such as copper (Cu), is being explored in clinical settings. Cu has long been known to have antimicrobial activity. However, Gram‐positive microorganisms, a class that includes pathogens commonly responsible for hospital‐acquired infection such as Staphylococcus aureus and Clostridioides difficile, are more resilient to its biocidal effect. Inspired by inherently bactericidal nanostructured surfaces found in nature, an improved Cu coating is developed, engineered to contain nanoscale surface features and thus increase its antibacterial activity against a broader range of organisms. In addition, a new method is established for facilitating the rapid and continuous release of biocidal metal ions from the coating, through incorporation of an antibacterial metal salt (ZnCl2) with a lower reduction potential than Cu. Electrophoretic deposition (EPD) is used to fabricate these coatings, which serves as a low‐cost and scalable route for modifying existing conductive surfaces with complex shape. By tuning both the surface morphology and chemistry, a nanocomposite Cu coating is created that decreases the microbial bioburden of Gram‐positive S. aureus by 94% compared to unmodified Cu.

The production of Necator americanus larvae for use in experimental human infection

BackgroundAlthough there is unprecedented interest in experimental human hookworm infection, details of hookworm manufacture and characterisation have been sparsely reported. In this report, we detail the production and characterisation of Necator americanus larvae for use in a recently published clinical trial.MethodsFaeces was obtained from an experimentally infected donor. Faecal hookworm DNA was determined by quantitative PCR. Paired samples were incubated in either sterile water or sterile water mixed with antimicrobials (amphotericin and gentamicin). Coproculture was performed by modified Harada-Mori method. The harvested larvae were then processed in either sterile water or antiseptic solution. Larval yield was then calculated (larvae per gram), larval viability was determined by thermally induced motility assay and microbial burden was determined at the day of harvest, at 48 h and at 7 days.ResultsTwenty-eight faecal cultures were performed over 16 months. The faecal hookworm DNA content was variable over this time. There was no association of larval yield with faecal hookworm DNA content. Pre-treatment of faeces with antimicrobials did not influence larval yield. Larval motility was 85.3% (95% CI 79.3–91.3%). Incubation of larvae in antiseptics did not reduce viability at 14 days with a marginal mean of 68.6% (95% CI 59.1–78.1%) washed in water vs. 63.3% (95% CI 53.8 – 72.9%) when incubated in betadine (p = 0.38). Larvae washed in sterile water did not meet microbial bioburden criteria. Incubation in antiseptic resulted in acceptable microbial bioburden at 48 h but not at 7 days. Although the addition of gentamicin did reduce the microbial bio-burden acceptable levels, it was found to significantly lower larval motility at 7 days compared to incubation in sterile water and motility at 7 days 37.8% (95% CI 4.7–70.9%) vs. 67.3% (95% CI 35.2–99.3%, p < 0.001), respectively.ConclusionsDespite standardised culture methodologies and the use of a single donor, larval yield varied considerably between batches and had no association with faecal hookworm DNA. Larval viability decreases over time and the age of larvae at time of use are likely to be important. Microbial bioburden maybe temporarily reduced by incubation in antiseptics and has little effect on viability. Incubation of larvae in gentamicin is effective at reducing microbial bioburden but is deleterious to larval viability.Graphical