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Objective: Although needleless connectors (NCs) are widely used in clinical practice, they carry significant risk of bloodstream infection (BSI). In this study, we quantified differences in bacterial transfer and biofilm formation between various NCs. Design: Prospective, clinically simulated in vitro experimental study. Methods: We tested 20 NCs in a 5-day clinical simulation of Staphylococcus aureus inoculations onto NC septum surfaces, which were then flushed with saline and cultured for bacterial transfer. Biofilm formation was measured through destructive sampling of the connector-catheter system. Moreover, 8 NC design factors were evaluated for their influence on bacterial transfer and biofilm formation. This study was designed without a disinfection protocol to ascertain the intrinsic risk of each NC. Results: Clave Neutron and MicroClave had the lowest overall mean log density of bacteria in the flush compared to other NCs (P < .05), except there were no statistically significant differences between Clave Neutron, Microclave, SafeTouch, and SafeAccess (P ≥ .05). The amount of biofilm in the NC was positively associated with bacteria in the flush (P < .0005). Among 8 design factors, flow path was most important, with the internal cannula associated with a statistically significant 1 log reduction (LR) in bacteria in the flush (R2 = 49%) and 0.5–2 LR in the connector (R2 = 34%). All factors together best explained bacteria in the flush (R2 = 65%) and biofilm in the connector (R2 = 48%). Conclusions: Bacterial transfer and biofilm formation in the connector-catheter system varied statistically significantly between the 20 NCs, suggesting that NC choice can lower the risk of developing catheter-related BSIs.

Background There is emerging evidence that implementation of an automated hand hygiene monitoring system (AHHMS) must be part of a multimodal hand hygiene (HH) program that includes complementary strategies. There are few published studies describing in detail the intervention strategies used with an AHHMS. Methods An AHHMS that provides group HH performance rates (100 x HH product dispenses divided by the number of room entries plus exits) was implemented on two Acute Care (AC) units and six long-term care (LTC) units at a Veterans Affairs Medical Center from March 2021 through April 2022. After a 4-week baseline period and 2.5-week washout period, the 52-week intervention period included many components, such as weekly huddles, unit nurse manager engagement, vendor provided clinician-based training and feedback, leadership support, unit recognition, signage and development of a new slogan to remind colleagues to perform HH. Statistical analysis was performed with a Poisson general additive mixed model. Results During the 4-week baseline period, the median HH performance rate was 18.6 (95% CI: [16.5, 21.0]) for all 8 units. During the intervention period, the median HH rate increased to 21.6 [19.1, 24.4], and during the last 4 weeks of the intervention period (exactly 1 year after baseline), the 8 units exhibited a median HH rate of 25.1 [22.2, 28.4], (p < 0.0001) [Figure 1]. The median HH rate increased from 17.5 to 20.0 (p < 0.0001) in LTC units and from 22.9 to 27.2 (p < 0.0001) in AC units. The intervention increased the use of hand sanitizer from 57.5% during baseline to 65.1% (p < 0.0001). The increase in HH rates was due to HH events increasing from 88,758 dispenses during the baseline to 123,722 dispenses during the last 4 weeks of the intervention. Direct observation results during the same periods showed HH compliance ranging from 61-86%. Figure 1- Monthly Hand Hygiene Performance Rates for all Units The green curve shows the change in the median HH rate during the intervention period compared to the baseline and washout periods, with vertical bars showing 95% confidence intervals for the monthly HH rate. Conclusion The intervention increased hand sanitizer usage and HH performance rates for all units. AC units were consistently better than LTC units, which have more visitors and more mobile veterans. Further HH improvement will rely on continued implementation of complementary strategies and long-term monitoring. Disclosures James W. Arbogast, PhD, GOJO Industries, Inc.: employee Pamela Wagner, RN MSN CPPS, GOJO Industries, Inc: Employee of GOJO Gregory A. Robbins, BS, GOJO Industries, Inc.: Current employee of GOJO Industries, Inc. Albert E. Parker, PhD, GOJO Industries: Advisor/Consultant John M. Boyce, MD, Diversey: Advisor/Consultant|Diversey: Expert Testimony|Diversey: Travel support|GOJO Industries: Advisor/Consultant|GOJO Industries: Expert Testimony|GOJO Industries: Travel support|Sodexo Healthcare: Advisor/Consultant.

Urea-hydrolysing biofilms are crucial to applications in medicine, engineering, and science. Quantitative information about ureolysis rates in biofilms is required to model these applications. We f...

Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the surface and disaggregating the clumps, and analyzing the sample. Of the four steps, harvesting and disaggregation are the least studied but nonetheless critical when considering the potential for test bias. This article demonstrates commonly used harvesting and disaggregation techniques for biofilm grown on three different surfaces. The three biofilm harvesting and disaggregation techniques, gleaned from an extensive literature review, include vortexing and sonication, scraping and homogenization, and scraping, vortexing and sonication. Two surface types are considered: hard non-porous (polycarbonate and borosilicate glass) and porous (silicone). Additionally, we provide recommendations for the minimum information that should be included when reporting the harvesting technique followed and an accompanying method to check for bias.

Metagenomic studies have revolutionized our understanding of the metabolic potential of uncultured microorganisms in various ecosystems. However, many of these genomic predictions have yet to be experimentally tested, and the functional expression of genomic potential often remains unaddressed. In order to obtain a more thorough understanding of cell physiology, novel techniques capable of testing microbial metabolism under close to in situ conditions must be developed. Here, we provide a benchmark study to demonstrate that bioorthogonal non-canonical amino acid tagging (BONCAT) in combination with fluorescence-activated cell sorting (FACS) and 16S rRNA gene sequencing can be used to identify anabolically active members of a microbial community incubated in the presence of various growth substrates or under changing physicochemical conditions. We applied this approach to a hot spring sediment microbiome from Yellowstone National Park (Wyoming, USA) and identified several microbes that changed their activity levels in response to substrate addition, including uncultured members of the phyla Thaumarchaeota, Acidobacteria, and Fervidibacteria. Because shifts in activity in response to substrate amendment or headspace changes are indicative of microbial preferences for particular growth conditions, results from this and future BONCAT-FACS studies could inform the development of cultivation media to specifically enrich uncultured microbes. Most importantly, BONCAT-FACS is capable of providing information on the physiology of uncultured organisms at as close to in situ conditions as experimentally possible.

Purpose Infusate osmolarity, pH, and cytotoxicity were investigated as risk factors for midline catheter failure. Methods An experimental, randomized, controlled, blinded trial was conducted using an ovine model. Two 10-cm, 18-gauge single-lumen midline catheters were inserted into the cephalic veins of sheep. The animals were divided into 6 study arms and were administered solutions of vancomycin 4 mg/mL (a low-cytotoxicity infusate) or 10 mg/mL (a high-cytotoxicity infusate), doxycycline 1 mg/mL (an acidic infusate), or acyclovir 3.5 mg/mL (an alkaline infusate) and 0.9% sodium chloride injection; or 1 of 2 premixed Clinimix (amino acids in dextrose; Baxter International) products with respective osmolarities of 675 mOsm/L (a low-osmolarity infusate) and 930 mOsm/L (a mid-osmolarity infusate). Contralateral legs were infused with 0.9% sodium chloride injection for control purposes. Catheter failure was evaluated by assessment of adverse clinical symptoms (swelling, pain, leakage, and occlusion). A quantitative vessel injury score (VIS) was calculated by grading 4 histopathological features: inflammation, mural thrombus, necrosis, and perivascular reaction. Results Among 20 sheep included in the study, the overall catheter failure rate was 95% for test catheters (median time to failure, 7.5 days; range, 3–14 days), while 60% of the control catheters failed before or concurrently (median time to failure, 7 days; range, 4.5–14 days). Four of the 6 study arms (all but the Clinimix 675-mOsm/L and acyclovir 3.5-mg/mL arms) demonstrated an increase in mean VIS of ≥77% in test vs control legs (P ≤ 0.034). Both pain and swelling occurred at higher rates in test vs control legs: 65% vs 10% and 70% vs 50%, respectively. The mean difference in rates of occlusive pericatheter mural thrombus between the test and control arms was statistically significant for the vancomycin 10-mg/mL (P = 0.0476), Clinimix 930-mOsm/L (P = 0.0406), and doxycycline 1-mg/mL (P = 0.032) arms. Conclusion Administration of infusates of varied pH, osmolarity, and cytotoxicity via midline catheter resulted in severe vascular injury and premature catheter failure; therefore, the tested infusates should not be infused via midline catheters.

In microbial studies, samples are often treated under different experimental conditions and then tested for microbial survival. A technique, dating back to the 1880s, consists of diluting the samples several times and incubating each dilution to verify the existence of microbial colony-forming units or CFU’s, seen by the naked eye. The main problem in the dilution series data analysis is the uncertainty quantification of the simple point estimate of the original number of CFU’s in the sample (i.e., at dilution zero). Common approaches such as log-normal or Poisson models do not seem to handle well extreme cases with low or high counts, among other issues. We build a novel binomial model, based on the actual design of the experimental procedure including the dilution series. For repetitions, we construct a hierarchical model for experimental results from a single laboratory and in turn a higher hierarchy for inter-laboratory analyses. Results seem promising, with a systematic treatment of all data cases, including zeros, censored data, repetitions, intra- and inter-laboratory studies. Using a Bayesian approach, a robust and efficient MCMC method is used to analyze several real data sets.

Aim To assess removal versus kill efficacies of antimicrobial treatments against thick biofilms with statistical confidence. Methods and results A photo-activated chlorine dioxide treatment (Photo ClO2) was tested in two independent experiments against thick (>100 μm) Pseudomonas aeruginosa biofilms. Kill efficacy was assessed by viable plate counts. Removal efficacy was assessed by 3D confocal scanning laser microscope imaging (CSLM). Biovolumes were calculated using an image analysis approach that models the penetration limitation of the laser into thick biofilms using Beer's Law. Error bars are provided that account for the spatial correlation of the biofilm's surface. The responsiveness of the biovolumes and plate counts to the increasing contact time of Photo ClO2 were quite different, with a massive 7 log reduction in viable cells (95% confidence interval [CI]: 6.2, 7.9) but a more moderate 73% reduction in biovolume (95% CI: [60%, 100%]). Results are leveraged to quantitatively assess candidate CSLM experimental designs of thick biofilms. Conclusions Photo ClO2 kills biofilm bacteria but only partially removes the biofilm from the surface. To maximize statistical confidence in assessing removal, imaging experiments should use fewer pixels in each z-slice, and more importantly, at least two independent experiments even if there is only a single field of view in each experiment. Significance and impact of study There is limited penetration depth when collecting 3D confocal images of thick biofilms. Removal can be assessed by optimally fitting Beer's Law to all of the intensities in a 3D image and by accounting for the spatial correlation of the biofilm's surface. For thick biofilms, other image analysis approaches are biased or do not provide error bars. We generate unbiased estimates of removal and assess candidate CSLM experimental designs of thick biofilms with different pixilations, numbers of fields of view and number of experiments using the included design tool.

Microtiter plate methods are commonly used for biofilm assessment. However, results obtained with these methods have often been difficult to reproduce. Hence, it is important to obtain a better understanding of the repeatability and reproducibility of these methods. An interlaboratory study was performed in five different laboratories to evaluate the reproducibility and responsiveness of three methods to quantify Staphylococcus aureus biofilm formation in 96-well microtiter plates: crystal violet, resazurin, and plate counts. An inter-lab protocol was developed for the study. The protocol was separated into three steps: biofilm growth, biofilm challenge, biofilm assessment. For control experiments participants performed the growth and assessment steps only. For treatment experiments, all three steps were performed and the efficacy of sodium hypochlorite (NaOCl) in killing S. aureus biofilms was evaluated. In control experiments, on the log10-scale, the reproducibility SD (SR) was 0.44 for crystal violet, 0.53 for resazurin, and 0.92 for the plate counts. In the treatment experiments, plate counts had the best responsiveness to different levels of efficacy and also the best reproducibility with respect to responsiveness (Slope/SR = 1.02), making it the more reliable method to use in an antimicrobial efficacy test. This study showed that the microtiter plate is a versatile and easy-to-use biofilm reactor, which exhibits good repeatability and reproducibility for different types of assessment methods, as long as a suitable experimental design and statistical analysis is applied.

In the food industry, the increasing antimicrobial resistance of food-borne pathogens to conventional sanitizers poses the risk of food contamination and a decrease in product quality and safety. Therefore, we explored alternative antimicrobials N-Acetyl-l-cysteine (NAC), rhamnolipids (RLs), and usnic acid (UA) as a novel approach to prevent biofilm formation and reduce existing biofilms formed by important food-borne pathogens (three strains of Salmonella enterica and two strains of Escherichia coli, Listeria monocytogenes, Staphylococcus aureus). Their effectiveness was evaluated by determining minimum inhibitory concentrations needed for inhibition of bacterial growth, biofilm formation, metabolic activity, and biofilm reduction. Transmission electron microscopy and confocal scanning laser microscopy followed by image analysis were used to visualize and quantify the impact of tested substances on both planktonic and biofilm-associated cells. The in vitro cytotoxicity of the substances was determined as a half-maximal inhibitory concentration in five different cell lines. The results indicate relatively low cytotoxic effects of NAC in comparison to RLs and UA. In addition, NAC inhibited bacterial growth for all strains, while RLs showed overall lower inhibition and UA inhibited only the growth of Gram-positive bacteria. Even though tested substances did not remove the biofilms, NAC represents a promising tool in biofilm prevention.

Background: Athletic training rooms have a high prevalence of bacteria, including multidrug-resistant organisms, increasing the risk for both local and systematic infections in athletes. There are limited data outlining formal protocols or standardized programs to reduce bacterial and viral burden in training rooms as a means of decreasing infection rate at the collegiate and high school levels. Hypothesis: Adaptation of a hygiene protocol would lead to a reduction in bacterial and viral pathogen counts in athletic training rooms. Study Design: Cohort study. Level of Evidence: Level 3. Methods: Two high school and 2 collegiate athletic training rooms were studied over the course of the 2017-2018 academic year. A 3-phase protocol, including introduction of disinfectant products followed by student-athlete and athletic trainer education, was implemented at the 4 schools. Multiple surfaces in the athletic training rooms were swabbed at 4 time points throughout the investigation. Bacterial and viral burden from swabs were analyzed for overall bacterial aerobic plate count (APC), bacterial adenosine triphosphate activity, influenza viral load, and multidrug-resistant organisms such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE). Results: Overall bacterial load, as measured by APC, was reduced by 94.7% (95% CI, 72.6-99.0; P = 0.003) over the course of the investigation after protocol implementation. MRSA and VRE were found on 24% of surfaces prior to intervention and were reduced to 0% by the end of the study. Influenza was initially detected on 25% of surfaces, with no detection after intervention. No cases of athletic training room–acquired infections were reported during the study period. Conclusion: A uniform infection control protocol was effective in reducing bacterial and viral burden, including multidrug-resistant organisms, when implemented in the athletic training rooms of 2 high schools and 2 colleges. Clinical Relevance: A standardized infection control protocol can be utilized in athletic training rooms to reduce bacterial and viral burden.

Background:The effectiveness of alcohol-based hand rub (ABHR) is correlated with drying time, which depends on the volume applied. Evidence suggests that there is considerable variation in the amount of ABHR used by healthcare providers.Objective:We sought to identify the volume of ABHR preferred for use by nurses.Methods:A prospective observation study was performed in 8 units at a tertiary-care hospital. Nurses were provided pocket-sized ABHR bottles with caps to record each bottle opening. Nurses were instructed to use the volume of ABHR they felt was best. The average ABHR volume used per hand hygiene event was calculated using cap data and changes in bottle mass.Results:In total, 53 nurses participated and 140 nurse shifts were analyzed. The average ABHR dose was 1.09 mL. This value was greater for non-ICU nurses (1.18 mL) than ICU nurses (0.96 mL), but this difference was not significant. We detected no significant association between hand surface area and preferred average dose volume. The ABHR dose volume was 0.006 mL less per use as the number of applications per shift increased (P = .007).Conclusions:The average dose of ABHR used was similar to the dose provided by the hospital’s automated dispensers, which deliver 1.1 mL per dose. The volume of ABHR dose was inversely correlated with the number of applications of ABHR per shift and was not correlated with hand size. Further research to understand differences and drivers of ABHR volume preferences and whether automated ABHR dosing may create a risk for people with larger hands is warranted.

Microbial contamination in metalworking systems is a critical problem. This study determined the microbial communities in metalworking fluids (MWFs) from two machining shops and investigated the effect of quorum sensing inhibition (QSI) on biofilm growth. In both operations, biofilm-associated and planktonic microbial communities were dominated by Pseudomonadales (60.2–99.7%). Rapid recolonization was observed even after dumping spent MWFs and meticulous cleaning. Using Pseudomonas aeruginosa PAO1 as a model biofilm organism, patulin (40 µM) and furanone C-30 (75 µM) were identified as effective QSI agents. Both agents had a substantially higher efficacy compared to α-amylase (extracellular polymeric substance degrading enzyme) and reduced biofilm formation by 63% and 76%, respectively, in MWF when compared to untreated controls. Reduced production of putatively identified homoserine lactones and quinoline in MWF treated with QS inhibitors support the effect of QSI on biofilm formation. The results highlight the effectiveness of QSI as a potential strategy to eradicate biofilms in MWFs.

Rapid testing methods can identify outbreaks and trigger preventive strategies for slowing the spread of SARS-CoV-2, the virus that causes COVID-19. The “gold-standard” detection method for SARS-CoV-2 is reverse transcription quantitative polymerase chain reaction (RT-qPCR) performed on samples collected using a nasopharyngeal (NP) swab. While NP RT-qPCR provides high sensitivity, it requires trained personnel to administer and suffers from lengthy time-to-result. Recently, the testing community has turned to rapid saliva-based screening methods including saliva-to-RT-qPCR and/or saliva-to-RT-LAMP (reverse transcription loop-mediated isothermal amplification) to identify infected individuals regardless of symptomatic presentation. Here, we report a simple and rapid RT-LAMP fluorometric assay performed directly on heat-inactivated saliva, without the addition of buffers or proteinase K treatments we call saliva LAMP (SLAMP). Over the course of two days, a total of 243 individuals were tested using NP RT-qPCR, saliva-based qPCR, and saliva-based RT-LAMP. Of the 243 NP RT-qPCR tests, 65 were positive, 178 were negative, and SLAMP demonstrated a 91% sensitivity and 98% specificity. SLAMP sensitivity becomes 95% when samples negative in saliva tests while positive in NP RT-qPCR are excluded from evaluation, potentially indicating significant differences in viral titer between collection sites on the body. SLAMP is performed in triplicates and takes 45 min to run in the laboratory, requiring less technician time and instrument run time than NP RT-qPCR. These results demonstrate that saliva-based RT-LAMP can enable frequent and rapid screening of large numbers of people to identify pre-symptomatic and asymptomatic individuals thereby controlling outbreaks.

Final publication is available from Mary Ann Liebert, Inc., publishers https://dx.doi.org/10.1089/ast.2021.0021. Deposited by shareyourpaper.org and openaccessbutton.org. We've taken reasonable steps to ensure this content doesn't violate copyright. However, if you think it does you can request a takedown by emailing help@openaccessbutton.org.

The development, validation, and use of new quantitative methodologies for testing the effectiveness of antimicrobial products are necessary to meet the regulatory challenges associated with an ever-changing marketplace, novel product claims, new infection control practices, and the emergence of new clinical pathogens. A series of four interlaboratory studies were conducted in a standardized manner on an interim quantitative method for testing liquid treatments against bacteria to assess its statistical performance. The Quantitative Method, a derivative of ASTM E2197, is designed to enumerate the number of viable microbes remaining on a test carrier following exposure to a liquid antimicrobial treatment; a log

Beer's Law explains how light attenuates into thick specimens, including thick biofilms. We use a Bayesian optimality criterion, the maximum of the posterior probability distribution, and computationally efficiently fit Beer's Law to the 3D intensity data collected from thick living biofilms by a confocal scanning laser microscope. Using this approach the top surface of the biofilm and an optimal image threshold can be estimated. Biofilm characteristics, such as bio-volumes, can be calculated from this surface. Results from the Bayesian approach are compared to other approaches including the method of maximum likelihood or simply counting bright pixels. Uncertainty quantification (i.e., error bars) can be provided for the parameters of interest. This approach is applied to confocal images of stained biofilms of a common lab strain of Pseudomonas aeruginosa, stained biofilms of Janthinobacterium isolated from the Antarctic, and biofilms of Staphylococcus aureus that have been genetically modified to fluoresce green.

The publisher's final version of this work can be found at https://doi.org/10.1137/18M1234096. Deposited by shareyourpaper.org and openaccessbutton.org. We've taken reasonable steps to ensure this content doesn't violate copyright. However, if you think it does you can request a takedown by emailing help@openaccessbutton.org.

Laboratory biofilm reactors are tools that researchers use to grow biofilms that exhibit characteristics sufficiently similar to the environment of interest. Numerous biofilm reactors that model various fluid dynamics are described in scientific literature, each with its associated list of advantages and limitations. This chapter focuses on the process used to design and fabricate biofilm reactors with the stated goal of generating a commercial product. The process begins with identifying the environment of interest and key attributes the reactor should include or model. A prototype is then designed, built, and tested in the laboratory. Modifications are made based upon laboratory performance until a design is achieved that is affordable, practical, operationally simple, and relevant and that provides repeatable, convincing results. This process was used to design the industrial surfaces biofilm reactor, developed to model cooling tower biofilms but suitable to study biofilms grown under low shear, high gas transfer, and intermittently wet conditions.

The inactivation of Pseudomonas aeruginosa biofilms grown on glass under high shear stress and exposed to a range of dissolved ozone concentrations (2, 5 and 7 ppm) at 10 and 20 min was investigated. The regression equation, log reduction (biofilm) = 0.64 + 0.59×(C – 2) + 0.33×(T – 10), described the dependence of biofilm inactivation on the dissolved ozone concentration (C, ppm) and contact time (T, min). The predicted D-values were 11.1, 5.7 and 2.2 min at 2, 5 and 7 ppm, respectively. Inactivation of biofilms grown on various surfaces was tested at a single dissolved ozone concentration of 5 ppm and a single exposure time of 20 min. Biofilms grown on plastic materials showed inactivation results similar to that of biofilms on glass, while biofilms grown on ceramics were statistically significantly more difficult to inactivate, suggesting the importance of utilizing non-porous materials in industrial and clinical settings.

Three dimensional confocal scanning laser microscope images offer dramatic visualizations of the action of living biofilms before and after interventions. Here we use confocal microscopy to study the effect of a treatment over time that causes a biofilm to swell and contract due to osmotic pressure changes. From these data, our goal is to reconstruct biofilm surfaces, to estimate the effect of the treatment on the biofilm’s volume, and to quantify the related uncertainties. We formulate the associated massive linear Bayesian inverse problem and then solve it using iterative samplers from large multivariate Gaussians that exploit well-established polynomial acceleration techniques from numerical linear algebra. Because of a general equivalence with linear solvers, these polynomial accelerated iterative samplers have known convergence rates, stopping criteria, and perform well in finite precision. An explicit algorithm is provided, for the first time, for an iterative sampler that is accelerated by the synergistic implementation of preconditioned conjugate gradient and Chebyshev polynomials.

Dissolved organic matter (DOM) in freshwater environments is an important source of organic carbon, supporting bacterial respiration. Frozen environments cover vast expanses of our planet, with glaciers and ice‐sheets storing upwards of 6 petagrams of organic carbon. It is generally believed that DOM liberated from ice stimulates downstream environments. If true, glacial DOM is an important component of global carbon cycling. However, coupling the release of DOM to microbial activity is challenging due to the molecular complexity of DOM and the metabolic connectivity within microbial communities. Using a single environmentally relevant organism, we demonstrate that processing of compositionally diverse DOM occurs, but, even though glacially derived DOM is chemically labile, it is unable to support sustained respiration. In view of projected changes in glacier DOM export, these findings imply that biogeochemical impacts on downstream environments will depend on the reactivity and heterogeneity of liberated DOM, as well as the timescale.

Slow rates of coal-to-methane conversion limit biogenic methane production from coalbeds. This study demonstrates that rates of coal-to-methane conversion can be increased by the addition of small amounts of organic amendments. Algae, cyanobacteria, yeast cells, and granulated yeast extract were tested at two concentrations (0.1 and 0.5 g/L), and similar increases in total methane produced and methane production rates were observed for all amendments at a given concentration. In 0.1 g/L amended systems, the amount of carbon converted to methane minus the amount produced in coal only systems exceeded the amount of carbon added in the form of amendment, suggesting enhanced coal-to-methane conversion through amendment addition. The amount of methane produced in the 0.5 g/L amended systems did not exceed the amount of carbon added. While the archaeal communities did not vary significantly, the bacterial populations appeared to be strongly influenced by the presence of coal when 0.1 g/L of amendment was added; at an amendment concentration of 0.5 g/L the bacterial community composition appeared to be affected most strongly by the amendment type. Overall, the results suggest that small amounts of amendment are not only sufficient but possibly advantageous if faster in situ coal-to-methane production is to be promoted.

Background: Alcohol-based hand rubs (ABHRs) are the primary form of hand hygiene in healthcare settings globally. Many developed countries, and most US hospitals utilize wall-mounted ABHR dispensers throughout the facility. The adoption of automated touch-free dispensers is increasing. However, data on the efficacy of ABHRs when used at dispensed amounts are limited. The evidence is strong, showing that formulation matters (not just alcohol concentration) and that agent volume impacts efficacy. Objective: We evaluated the efficacy of ABHR foams on human hands using 2 controlled test methods at variable volumes (ie, typical doses and realistic volumes that healthcare personnel could use in patient care practice). Methods: We tested 8 commercially available ABHR products, the WHO hand-rub formulation, (P1–P9) and a nonantibacterial foam handwash control (P10) on human participants at 2 different application frequencies (“1 application” and “10 applications”) using 2 different ASTM test methods (E1174 and E2755). Studies using ASTM-E1174 evaluated 3 different application volumes (0.7 mL, 1.1 mL, and 2.0 mL) of the 10 products, each tested on 12–13 subjects. Studies using ASTM-E2755 evaluated a single 1.1 mL volume for the 9 ABHR products (P1–P9), each on 2–12 subjects. A linear mixed-effects model was fit separately to log reductions with random effects for subject and date, and a fixed effect for product. Results: Four different foam formulations (P1–P4) consistently outperformed all other formulations by the E1174 method, especially with increasing volumes and after 10 product applications (Fig. 1). When tested with E2755, all formulations performed similarly, with only P1 and P2 differentiating after 10 applications (Fig. 2). ABHR efficacy consistently increased with larger application volumes, whereas the handwash control (P10) achieved a similar efficacy (∼2 log reduction) at all volumes. Efficacy for some ABHR formulations increased, whereas others decreased with repeated applications. Alcohol concentration did not correlate with log reduction. Conclusions: Formulation and the product application volume affect the antimicrobial efficacy of ABHR; therefore, those data should be critically assessed by healthcare personnel assessing ABHR product performance. Test methods matter: when E1174 was used, greater differentiation between formulations was observed. This may be due to the larger contamination volume and greater soil load used in E1174.Funding: GOJO Industries, Inc., provided Funding: for this study.Disclosures: James W. Arbogast and David R. Macinga report salary from GOJO Industries.

Background: Technology and interest for use of automated hand hygiene monitoring systems (AHHMS) as a tool to help improve healthcare personnel hand hygiene has been advancing for the last decade. Emerging evidence indicates that the use of AHHMS plus complementary strategies improves hand hygiene (HH) performance rates and outcomes (eg, healthcare-associated infections). The WHO HH guideline “Multimodal Strategy” teaches the importance of multiple components as necessary to build and sustain HH compliance. Few published data compare the impact of different complementary behavioral strategies in combination with AHHMS on results. Methods: We utilized data from 1 AHHMS that records alcohol-based hand rub and soap dispensing and room entries and exits to provide group HH performance rates. Data were collected from 58 units in 10 hospitals in North America from July 2014 through August 2019. Hospitals were stratified into 4 categories based on their approach to hospital-initiated unit-level interventions and AHHMS vendor support (Table 1). Baseline data were defined for each unit as the initial 1–2 months of execution, before complementary strategies were initiated. Statistical analysis was performed on the annual number of dispenses and opportunities with a mixed-effects Poisson regression with random effects for facility, unit and year and fixed effects for intervention type and unit type. Interactions were not included in the model based on interaction plots and significance tests. Poisson assumptions were verified with Pearson residual plots. Results: HH performance rates overall and compared to the baseline are shown in Table 2. More than 8 million opportunities were achieved in all 58 units combined. An intervention strategy with multiple complementary components (ie, clinical support provided by the AHHMS vendor plus hospital-initiated unit level interventions) yielded significantly better HH performance than all other categories (>20% increase, P < .00001). Somewhat surprisingly, vendor clinical support or hospital-initiated, unit-level interventions alone with the AHHMS yielded a slight decrease in HH performance relative to AHHMS only (P < .00001). Conclusions: AHHMS is a useful tool in understanding HH performance and identifying unit-based initiatives that need attention. Implementation of an AHHMS by itself or with limited complementary behavior-change strategies does not drive improvement. Support provided by the vendor and hospital-initiated, complementary strategies were not sufficient additions to the AHHMS individually, but in combination they resulted in the greatest improvements in HH performance. These findings illustrate the value of a partnership between the hospital and the AHHMS vendor.Funding: GOJO Industries, Inc., provided support for this study.Disclosures: James W. Arbogast, Lori D. Moore and Megan DiGiorgio report salary from GOJO Industries.

Background: Hand hygiene (HH) has long been a focus in the prevention of healthcare-associated infections. The limitations of direct observation, including small sample size (often 20–100 observations per month) and the Hawthorne effect, have cast doubt on the accuracy of reported compliance rates. As a result, hospitals are exploring the use of automated HH monitoring systems (AHHMS) to overcome the limitations of direct observation and to provide a more robust and realistic estimation of HH behaviors. Methods: Data analyzed in this study were captured utilizing a group-based AHHMS installed in a number of North American hospitals. Emergency departments, overflow units, and units with Results: Over the study period, 222.7 million HHOs and 99 million HHEs were captured in the data set. There were an average of 18.7 beds per unit. The average number of HHOs per unit per day was 3,528, and the average number of HHEs per unit per day was 1,572. The overall median compliance rate was 35.2 (95% CI, 31.5%–39.3%). Unit-to-unit comparisons revealed some significant differences: compliance rates for medical-surgical units were 12.6% higher than for intensive care units (P < .0001). Conclusions: This is the largest HH data set ever reported. The results illustrate the magnitude of HHOs captured (3,528 per unit per day) by an AHHMS compared to that possible through direct observation. It has been previously suggested that direct observation samples between 0.5% to 1.7% of all HHOs. In healthcare, it is unprecedented for a patient safety activity that occurs as frequently as HH to not be accurately monitored and reported, especially with HH compliance as low as it is in this multiyear, multicenter study. Furthermore, hospitals relying on direct observation alone are likely insufficiently allocating and deploying valuable resources for improvement efforts based on the scant information obtained. AHHMSs have the potential to introduce a new era in HH improvement.Funding: GOJO Industries, Inc., provided support for this study.Disclosures: Lori D. Moore and James W. Arbogast report salary from GOJO.

Understanding microbial community structure and function within the subsurface is critical to assessing overall quality and maintenance of groundwater; however, the factors that determine microbial community assembly, structure, and function in groundwater systems and their impact on water quality remains poorly understood. In this study, three shallow wells (FW301, FW303, FW305) in a non-contaminated shallow aquifer in the ENIGMA-Oak Ridge Field Research Center (Oak Ridge, TN) were sampled approximately 3 times a week over a period of three months to measure changes in groundwater geochemistry and microbial diversity. It was expected that the sampled microbial diversity from two historic field wells (FW301, FW303) would be relatively stable, while diversity from a newer well (FW305) would be less stable over time. The wells displayed some degree of hydrochemical variability over time unique to each well, with FW303 being overall the most stable well and FW301 being the most dynamic based upon dissolved oxygen, conductivity, and nitrate. Community analysis via ss-rRNA paired-end sequencing and distribution-based clustering revealed higher OTU richness, diversity, and variability in groundwater communities of FW301 than the other two wells for diversity binned over all time points. Microbial community composition of a given well was on average > 50% dissimilar to any other well at a given time (days), yet, functional gene diversity as measured with GeoChip remained relatively constant. Similarities in community structure across wells were observed with respect to the presence of 20 shared bacterial groups in all samples in all wells, although at varying levels over the tested time period. Similarity percentage (SIMPER) analysis revealed that variability in FW301 was largely attributed to low abundance, highly-transient populations, while variability in the most hydrochemically stable well (FW303) was due to fluctuations in more highly abundant and frequently present taxa. Additionally, the youngest well FW305 showed a dramatic shift in community composition towards the end of the sampling period that was not observed in the other wells, suggesting possible succession events over time. Time-series analysis using vector auto-regressive models and Granger causality showed unique relationships between richness and geochemistry over time in each well. These results indicate temporally dynamic microbial communities over short time scales, with day-to-day population shifts in local community structure influenced by available source community diversity and local groundwater hydrochemistry.

Through a statistical meta-analysis of published data on antimicrobial efficacy against biofilms formed by two common bacterial species, it was concluded that the particular experimental method used is the most important factor determining the outcome of the test. An expected dose-response relationship (greater killing with higher doses or longer treatment times) was observed for data sets derived from a single method but was not observed when data from multiple studies using diverse methods were pooled. Method-specific properties such as the surface area/volume ratio, areal biofilm cell density, and microbial species were shown to influence quantitative measurements of biofilm killing. A better appreciation of the method characteristics that affect antibiofilm efficacy tests could aid decision-making related to investment in research and development and regulatory approvals for biofilm control strategies. The following recommendations are offered to those working in research and development related to biofilm control: (i) report the log reduction, surface area/volume ratio, and biofilm areal cell density; (ii) include data for a benchmark agent, making sure that this agent performs competitively at the dose tested; (iii) measure the dose-response relationship, i.e., make measurements at multiple treatment concentrations or dose durations; and (iv) use a standardized method in addition to research methods.

The mixed effects of temperature (20 °C, 25 °C and 30 °C), nitrate concentration (0.5 mM and 2.0 mM), pH buffer, and bicarbonate addition (trigger) on biomass growth and lipid accumulation were investigated in the environmental alga PW95 during batch experiments in standardized growth medium. PW95 was isolated from coal-bed methane production water and classified as a Chlamydomonas-like species by morphological characterization and phylogenetic analysis (18S, ITS, rbcL). A factorial experimental design tested the mixed effects on PW95 before and after nitrate depletion to determine a low cost, high efficiency combination of treatments for biomass growth and lipid accumulation. Results showed buffer addition affected growth for most of the treatments and bicarbonate trigger had no statistically significant effect on growth and lipid accumulation. PW95 displayed the highest growth rate and chlorophyll content at 30 °C and 2.0 mM nitrate and there was an inverse relation between biomass accumulation and lipid accumulation at the extremes of nitrate concentration and temperature. The combination of higher temperature (30 °C) and lower nitrate level (0.5 mM) without the use of a buffer or bicarbonate addition resulted in maximal daily biomass accumulation (5.30 × 106 cells/mL), high biofuel potential before and after nitrate depletion (27% and 20%), higher biofuel productivity (16 and 15 mg/L/d, respectively), and desirable fatty acid profiles (saturated and unsaturated C16 and C18 chains). Our results indicate an important interaction between low nitrate levels, temperature, and elevated pH for trade-offs between biomass and lipid production in PW95. This work serves as a model to approach and advance the study of physiological responses of novel microalgae to diverse culture conditions that mimic environmental changes for outdoor biofuel production. The most promising conditions for growth and biofuel production were identified for PW95 and this approach can be implemented for other microalgal production systems.

Tracheal intubation disrupts physiological homeostasis of secretion production and clearance, resulting in secretion accumulation within endotracheal tubes (ETTs). Novel in vitro and in vivo models were developed to specifically recapitulate the clinical manifestations of ETT occlusion. The novel Sharklet™ micropatterned ETT was evaluated, using these models, for the ability to reduce the accumulation of both bacterial biofilm and airway mucus compared to a standard care ETT. Novel ETTs with micropattern on the inner and outer surfaces were placed adjacent to standard care ETTs in in vitro biofilm and airway patency (AP) models. The primary outcome for the biofilm model was to compare commercially-available ETTs (standard care and silver-coated) to micropatterned for quantity of biofilm accumulation. The AP model's primary outcome was to evaluate accumulation of artificial airway mucus. A 24-h ovine mechanical ventilation model evaluated the primary outcome of relative quantity of airway secretion accumulation in the ETTs tested. The secondary outcome was measuring the effect of secretion accumulation in the ETTs on airway resistance. Micropatterned ETTs significantly reduced biofilm by 71% (p = 0.016) compared to smooth ETTs. Moreover, micropatterned ETTs reduced lumen occlusion, in the AP model, as measured by cross-sectional area, in distal (85%, p = 0.005), middle (84%, p = 0.001) and proximal (81%, p = 0.002) sections compared to standard care ETTs. Micropatterned ETTs reduced the volume of secretion accumulation in a sheep model of occlusion by 61% (p

Hot water sanitization is a common means to maintain microbial control in process equipment for industries where microorganisms can degrade product or cause safety issues. This study compared the hot water inactivation kinetics of planktonic and biofilm-associated Sphingomonas parapaucimobilis at temperatures relevant to sanitization processes used in the pharmaceutical industry, viz. 65, 70, 75, and 80°C. Biofilms exhibited greater resistance to hot water than the planktonic cells. Both linear and nonlinear statistical models were developed to predict the log reduction as a function of temperature and time. Nonlinear Michaelis-Menten modeling provided the best fit for the inactivation data. Using the model, predictions were calculated to determine the times at which specific log reductions are achieved. While ≥80°C is the most commonly cited temperature for hot water sanitization, the predictive modeling suggests that temperatures ≥75°C are also effective at inactivating planktonic and biofilm bacteria in timeframes appropriate for the pharmaceutical industry.

Objective: The aim of this study was to determine the efficacy of a multimodal hand hygiene intervention program in reducing health care insurance claims for hygiene preventable infections (eg, cold and influenza), absenteeism, and subjective impact on employees. Methods: A 13.5-month prospective, randomized cluster controlled trial was executed with alcohol-based hand sanitizer in strategic workplace locations and personal use (intervention group) and brief hand hygiene education (both groups). Four years of retrospective data were collected for all participants. Results: Hygiene-preventable health care claims were significantly reduced in the intervention group by over 20% (P

National Science Foundation under Grant No. 0628258; Defense University Research Instrumentation Program (DURIP, Contract Number: W911NF0510255); MSU Thermal Biology Institute from the NASA Exobiology Program (Project NAG5-8807)

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Models of microbial community dynamics generally rely on a sub-scale model for microbial metabolisms. In systems such as distributed multispecies communities like biofilms, where it is not reasonable to simplify to a small number of limiting substrates, tracking the large number of active metabolites likely requires measurement or estimation of large numbers of kinetic and regulatory parameters. Alternatively, a largely kinetics-free methodology is proposed combining cellular level constrained, steady state metabolic flux analysis with macro scale microbial community models. The methodology easily allows coupling of macroscale information, including measurement data, with cell-scale metabolism. Illustrative examples are included.

We review reproducibility results for methods that test antimicrobial efficacy against biofilms, spores and bacteria dried onto a surface. Our review, that included test results for Pseudomonas aeruginosa, Salmonella choleraesuis and Bacillus subtilis, suggests that the level of reproducibility depends on the efficacy of the antimicrobial agent being tested for each microbe and microbial environment. To determine the reproducibility of a method, several laboratories must independently test the same antimicrobial agent using the method. Little variability among the efficacy results suggests good reproducibility. Such reproducibility assessments currently are hampered by the absence of an objective process for deciding whether the variability is sufficiently small. We present a quantitative decision process that objectively determines whether any method that assesses antimicrobial efficacy is reproducible. Because the perception of acceptable reproducibility may differ among stakeholders, the decision process is governed by a stakeholder’s specifications that necessarily includes the efficacy of the agents to be tested.